Anti-cancer compounds and conjugates thereof

ABSTRACT

Disclosed herein are compounds, drug-conjugates thereof, methods of preparing drug-conjugates, and uses thereof. Also disclosed are pharmaceutical compositions and methods of treatment. The compounds and drug-conjugates disclosed herein can be used to treat a variety of conditions, diseases and ailments such as bladder cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, lung cancer, melanoma, non-Hodgkin lymphoma, glioblastoma, pancreatic cancer, prostate cancer, and thyroid cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/765,232, filed on Mar. 30, 2018, which is a U.S. National Phase ofInternational Application No. PCT/US2016/053991, filed on Sep. 27, 2016and published on Apr. 6, 2017 as WO 2017/058808, which claims thebenefit of U.S. Provisional Application No. 62/236,694, filed on Oct. 2,2015.

BACKGROUND Field

This application relates to the fields of chemistry and medicine, moreparticularly to anti-cancer compounds and drug-conjugates thereof,pharmaceutical compositions, and methods of treatment.

Description of the Related Technology

Recently, it has been found that an antibody (or antibody fragment suchas a single-chain variable fragment) can be linked to a payload drug toform an immunoconjugate that has been termed antibody-drug conjugate, orADC. The antibody causes the ADC to bind to the target cells. Bycombining the unique targeting capabilities of monoclonal antibodieswith the cancer-killing ability of cytotoxic drugs, antibody-drugconjugates allow sensitive discrimination between healthy and diseasedtissue. This means that, in contrast to traditional chemotherapeuticagents, antibody-drug conjugates target and attack the cancer cell sothat healthy cells are less severely affected.

In developing antibody-drug conjugates, an anticancer compound iscoupled to an antibody that specifically targets a specific marker (e.g.a protein that, ideally, is only to be found in or on tumor cells). TheADC may be absorbed or internalized in the target cell. After the ADC isinternalized, the cytotoxic compound may be released to provider ananticancer effect.

Despite the known benefits of ADCs, the highly toxic nature of knownpayload drugs still results in toxicity issues. For example, some freedrug is released systemically from the ADC and the ADC can accumulate inthe liver. Thus, there is a need for improved ADCs in which the payloadsremain relatively non-cytotoxic upon systemic free drug release,resulting in increased therapeutic windows.

Cytotoxic agents include drugs that are primarily used to treat cancer,frequently as part of a chemotherapy regime.

Cytotoxic agents have an effect of preventing the rapid growth anddivision of cancer cells. However, cytotoxic agents also affect thegrowth of other quick dividing cells in the body such as hair folliclesand the lining of the digestive system. As a result of the treatment,many normal cells are damaged along with the cancer cells.

Dolastatin 10 is a cytotoxic marine ascidian alkaloid having anticanceractivity, for example, in treating patients who have metastaticpancreatic cancer.

SUMMARY

Some embodiments provide compound-conjugates, methods of preparingcompound-conjugates, and uses thereof.

Some embodiments provide a compound having the structure of Formula I:

-   -   or pharmaceutically acceptable salts or solvents thereof,        wherein:    -   R¹ is selected from the group consisting of hydrogen, deuterium,        an optionally substituted C₁-C₆ alkyl, an optionally substituted        C₃-C₇ cycloalkyl, an optionally substituted aryl, and an        optionally substituted heteroaryl;    -   R² is selected from the group consisting of hydrogen, deuterium,        an optionally substituted C₁-C₆ alkyl, an optionally substituted        C₃-C₇ cycloalkyl, an optionally substituted aryl, and an        optionally substituted heteroaryl;    -   R³ is selected from the group consisting of hydrogen, deuterium,        and optionally substituted C₁-C₆ alkyl, an optionally        substituted hydroxyl, an optionally substituted C₁-C₆ alkoxy, an        optionally substituted C₃-C₇ cycloalkyl, an optionally        substituted aryl, and an optionally substituted heteroaryl;    -   R⁴ is an optionally substituted C₁-C₆ alkyl;    -   R⁵ is hydrogen, deuterium or an optionally substituted C₁-C₆        alkyl;    -   R⁶ is hydrogen, deuterium or an optionally substituted C₁-C₆        alkyl;    -   X¹ and X² are each independently selected from the group        consisting of hydrogen, deuterium, an optionally substituted        C₁-C₆ alkyl, an optionally substituted aryl, an optionally        substituted heteroaryl, halogen, —CN, —N₃, —COOR^(B),        —NR^(A)R^(B), —OR^(B), and —SR^(B), where at least one of X¹ and        X² is not hydrogen when R⁵ is methyl;    -   R^(A) is selected from the group consisting of hydrogen,        deuterium, an optionally substituted C₁-C₆ alkyl, an optionally        substituted C₃-C₇ cycloalkyl, an optionally substituted aryl,        and an optionally substituted heteroaryl;    -   R^(B) is selected from the group consisting of hydrogen,        deuterium, an optionally substituted C₁-C₆ alkyl, an optionally        substituted C₃-C₇ cycloalkyl, an optionally substituted aryl,        and an optionally substituted heteroaryl;    -   R⁷ is an optionally substituted C₁-C₆ alkyl, an optionally        substituted heteroaryl, or —C(═O)R^(C);    -   R^(C) is selected from the group consisting of an optionally        substituted C₁-C₆ alkyl, optionally substituted C₁-C₆ alkoxy, an        optionally substituted C₃-C₇ cycloalkyl, an optionally        substituted aryl, and an optionally substituted heteroaryl, and        hydroxyl; and    -   R⁸ is hydrogen, deuterium or hydroxyl.

Some embodiments provide a compound having the structure of Formula

-   -   or pharmaceutically acceptable salts or solvates thereof,    -   wherein:    -   R¹ is selected from the group consisting of an optionally        substituted C₁-C₆ alkyl, an optionally substituted C₃-C₇        cycloalkyl, an optionally substituted aryl, and an optionally        substituted heteroaryl;    -   R² is selected from the group consisting of R^(D), an optionally        substituted C₁-C₆ alkyl, an optionally substituted C₃-C₇        cycloalkyl, an optionally substituted aryl, and an optionally        substituted heteroaryl;    -   R³ is selected from the group consisting of hydrogen, deuterium,        and optionally substituted C₁-C₆ alkyl, an optionally        substituted hydroxyl, an optionally substituted C₁-C₆ alkoxy, an        optionally substituted C₃-C₇ cycloalkyl, an optionally        substituted aryl, and an optionally substituted heteroaryl;    -   R⁴ is an optionally substituted C₁-C₆ alkyl;    -   R⁵ is hydrogen, deuterium or an optionally substituted C₁-C₆        alkyl;    -   R⁶ is hydrogen, deuterium or an optionally substituted C₁-C₆        alkyl;    -   X¹ and X² are each independently selected from the group        consisting of hydrogen, deuterium, an optionally substituted        C₁-C₆ alkyl, an optionally substituted aryl, an optionally        substituted heteroaryl, halogen, —CN, —N₃, —COOR^(B),        —NR^(A)R^(B), —OR^(B), and —SR^(B), where at least one of X¹ and        X² is not hydrogen when R⁵ is methyl;    -   R^(A) is selected from the group consisting of hydrogen,        deuterium, an optionally substituted C₁-C₆ alkyl, an optionally        substituted C₃-C₇ cycloalkyl, an optionally substituted aryl,        and an optionally substituted heteroaryl;    -   R^(B) is selected from the group consisting of hydrogen,        R^(1A)-L, R^(3A)-L, R^(4A)-L¹-, Mc-L¹-, Mal-L³-L¹-,        R^(1A)-Mal-L³-L¹-Val-Cit-PAB—C(O)— or        Mal-L³-L¹-Val-Cit-PAB—C(O)—;    -   R⁷ is an optionally substituted C₁-C₆ alkyl, an optionally        substituted heteroaryl, or —C(═O)R^(C);    -   R^(C) is selected from the group consisting of an optionally        substituted C₁-C₆ alkyl, optionally substituted C₁-C₆ alkoxy, an        optionally substituted C₃-C₇ cycloalkyl, an optionally        substituted aryl, and an optionally substituted heteroaryl, and        hydroxyl; and    -   R⁸ is hydrogen, deuterium, —OR^(D), or —SR^(D);    -   R^(D) is selected from the group consisting of hydrogen,        R^(1A)—, R^(3A)-L¹-, R^(4A)-L¹-, Mc-L¹-, Mal-L³-L¹-,        R^(1A)-Mal-L³-L¹-Val-Cit-PAB—C(O)— and        Mal-L³-L¹-Val-Cit-PAB—C(O)—;    -   R^(3A) is

-   -   R^(4A) is a conjugation moiety;    -   R^(1A) is a conjugated targeting moiety;    -   L¹ is a linker or a bond; and    -   L³ is an alkanoyl.

Some embodiments provide a method of treating a disease selected fromthe group consisting of uterine sarcoma cancer, bladder cancer, breastcancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer,lung cancer, melanoma, non-Hodgkin lymphoma, leukemia, pancreaticcancer, prostate cancer, and thyroid cancer, comprising administering acompound as disclosed and described herein to a subject in need thereof.

Some embodiments provide a method delivering a compound having thestructure of Formula I to an in vivo mammalian cell, the methodcomprising administering a compound having the structure of Formula IIto a mammal comprising the in vivo mammalian cell.

Some embodiments provide use of a compound having the structure ofFormula II to provide a compound having the structure of Formula I to atarget location.

Also provided herein are the compounds described above conjugated to atargeting moiety with a linker. Also provided herein are the compoundsdescribed above with a linker.

DETAILED DESCRIPTION

Some embodiments provide a compound having the structure of Formula Iand/or II.

Some embodiments provide a composition including compound having thestructure of Formula I and/or II; and a carrier.

Definitions

As used herein, common organic abbreviations are defined as follows:

-   Ac Acetyl-   aq. Aqueous-   BOC or Boc tert-Butoxycarbonyl-   BrOP bromo tris(dimethylamino) phosphonium hexafluorophosphate-   Bu n-Butyl-   ° C. Temperature in degrees Centigrade-   DCM methylene chloride-   DEPC Diethylcyanophosphonate-   DIC diisopropylcarbodiimide-   DIEA Diisopropylethylamine-   DMF N,N-Dimethylformamide-   EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide-   Et Ethyl-   EtOAc Ethyl acetate-   Eq Equivalents-   Fmoc 9-Fluorenylmethoxycarbonyl-   g Gram(s)-   h Hour (hours)-   HATU 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium    hexafluorophosphate-   HOAt 1-Hydroxy-7-azabenzotriazole-   HOBT N-Hydroxybenzotriazole-   HOSu N-Hydroxysuccinimide-   HPLC High-performance liquid chromatography-   LC/MS Liquid chromatography-mass spectrometry-   Mal Maleimido-   Mc Maleimidocaproyl-   Me Methyl-   MeOH Methanol-   MeCN Acetonitrile-   mL Milliliter(s)-   MS mass spectrometry-   RP-HPLC reverse phase HPLC-   rt room temperature-   t-Bu tert-Butyl-   TBDPS tert-Butyldiphenylsilyl-   TEA Triethylamine-   Tert, t tertiary-   TFA Trifluoracetic acid-   THF Tetrahydrofuran-   THP Tetrahydropyranyl-   TLC Thin-layer chromatography-   μL Microliter(s)

The term “pharmaceutically acceptable salt” refers to salts that retainthe biological effectiveness and properties of a compound and, which arenot biologically or otherwise undesirable for use in a pharmaceutical.In many cases, the compounds disclosed herein are capable of formingacid and/or base salts by virtue of the presence of amino and/orcarboxyl groups or groups similar thereto. Pharmaceutically acceptableacid addition salts can be formed with inorganic acids and organicacids. Inorganic acids from which salts can be derived include, forexample, hydrochloric acid, hydrobromic acid, sulfuric acid, nitricacid, phosphoric acid, and the like. Organic acids from which salts canbe derived include, for example, acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinicacid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceuticallyacceptable base addition salts can be formed with inorganic and organicbases. Inorganic bases from which salts can be derived include, forexample, sodium, potassium, lithium, ammonium, calcium, magnesium, iron,zinc, copper, manganese, aluminum, and the like; particularly preferredare the ammonium, potassium, sodium, calcium and magnesium salts.Organic bases from which salts can be derived include, for example,primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, basic ionexchange resins, and the like, specifically such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine, andethanolamine. Many such salts are known in the art, as described in WO87/05297, Johnston et al., published Sep. 11, 1987 (incorporated byreference herein in its entirety).

As used herein, “C_(a) to C_(b)” or “C_(a)-b” in which “a” and “b” areintegers refer to the number of carbon atoms in the specified group.That is, the group can contain from “a” to “b”, inclusive, carbon atoms.Thus, for example, a “C₁ to C₄ alkyl” or “C-4 alkyl” group refers to allalkyl groups having from 1 to 4 carbons, that is, CH₃—, CH₃CH₂—,CH₃CH₂CH₂—, (CH₃)₂CH—, CH₃CH₂CH₂CH₂—, CH₃CH₂CH(CH₃)— and (CH₃)₃C—.

As used herein, “PAB” refers to a para-aminobenzyloxy moiety. The PABmoiety is typically depicted as a di-radical (i.e., has two points ofattachment to the rest of the molecule), it is to be understood that thePAB amino group provides an amide bond with the adjacent amino acidresidue (e.g. citrulline) and the PAB oxygen group provides a carbonatewith the adjacent oxycarbonyl or carbamate group with the adjacentaminocarbonyl of the depicted structure. Thus, for example, asubstituent depicted as -Cit-PAB—C(O)-Phe- has the following structure:

-   -   where the PAB moiety is shown within the brackets.

The term “halogen” or “halo,” as used herein, means any one of theradio-stable atoms of column 7 of the Periodic Table of the Elements,e.g., fluorine, chlorine, bromine, or iodine, with fluorine and chlorinebeing preferred.

As used herein, “alkyl” refers to a straight or branched hydrocarbonchain that is fully saturated (i.e., contains no double or triplebonds). The alkyl group may have 1 to 20 carbon atoms (whenever itappears herein, a numerical range such as “1 to 20” refers to eachinteger in the given range; e.g., “1 to 20 carbon atoms” means that thealkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbonatoms, etc., up to and including 20 carbon atoms, although the presentdefinition also covers the occurrence of the term “alkyl” where nonumerical range is designated). The alkyl group may also be a mediumsize alkyl having 1 to 9 carbon atoms. The alkyl group could also be alower alkyl having 1 to 4 carbon atoms. The alkyl group may bedesignated as “C₁₋₄ alkyl” or similar designations. By way of exampleonly, “C₁₋₄ alkyl” indicates that there are one to four carbon atoms inthe alkyl chain, i.e., the alkyl chain is selected from the groupconsisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl,sec-butyl, and t-butyl. Typical alkyl groups include, but are in no waylimited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiarybutyl, pentyl, hexyl, and the like.

As used herein, “alkoxy” refers to the formula —OR wherein R is an alkylas is defined above, such as “C-9 alkoxy”, including but not limited tomethoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy,iso-butoxy, sec-butoxy, and tert-butoxy, and the like.

As used herein, “alkylthio” refers to the formula —SR wherein R is analkyl as is defined above, such as “C₁₋₉ alkylthio” and the like,including but not limited to methylmercapto, ethylmercapto,n-propylmercapto, 1-methylethylmercapto (isopropylmercapto),n-butylmercapto, iso-butylmercapto, sec-butylmercapto,tert-butylmercapto, and the like.

As used herein, “alkenyl” refers to a straight or branched hydrocarbonchain containing one or more double bonds. The alkenyl group may have 2to 20 carbon atoms, although the present definition also covers theoccurrence of the term “alkenyl” where no numerical range is designated.The alkenyl group may also be a medium size alkenyl having 2 to 9 carbonatoms. The alkenyl group could also be a lower alkenyl having 2 to 4carbon atoms. The alkenyl group may be designated as “C₂₄ alkenyl” orsimilar designations. By way of example only, “C₂₋₄ alkenyl” indicatesthat there are two to four carbon atoms in the alkenyl chain, i.e., thealkenyl chain is selected from the group consisting of ethenyl,propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl,buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl,1-ethyl-ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl,buta-1,2-dienyl, and buta-1,2-dien-4-yl. Typical alkenyl groups include,but are in no way limited to, ethenyl, propenyl, butenyl, pentenyl, andhexenyl, and the like.

As used herein, “alkynyl” refers to a straight or branched hydrocarbonchain containing one or more triple bonds. The alkynyl group may have 2to 20 carbon atoms, although the present definition also covers theoccurrence of the term “alkynyl” where no numerical range is designated.The alkynyl group may also be a medium size alkynyl having 2 to 9 carbonatoms. The alkynyl group could also be a lower alkynyl having 2 to 4carbon atoms. The alkynyl group may be designated as “C₂₄ alkynyl” orsimilar designations. By way of example only, “C₂₋₄ alkynyl” indicatesthat there are two to four carbon atoms in the alkynyl chain, i.e., thealkynyl chain is selected from the group consisting of ethynyl,propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl, and2-butynyl. Typical alkynyl groups include, but are in no way limited to,ethynyl, propynyl, butynyl, pentynyl, and hexynyl, and the like.

The term “aromatic” refers to a ring or ring system having a conjugatedpi electron system and includes both carbocyclic aromatic (e.g., phenyl)and heterocyclic aromatic groups (e.g., pyridine). The term includesmonocyclic or fused-ring polycyclic (i.e., rings which share adjacentpairs of atoms) groups provided that the entire ring system is aromatic.

As used herein, “aryloxy” and “arylthio” refers to RO— and RS—, in whichR is an aryl as is defined above, such as “C₆₋₁₀ aryloxy” or “C₆₋₁₀arylthio” and the like, including but not limited to phenyloxy.

An “aralkyl” or “arylalkyl” is an aryl group connected, as asubstituent, via an alkylene group, such as “C₇₋₁₄ aralkyl” and thelike, including but not limited to benzyl, 2-phenylethyl,3-phenylpropyl, and naphthylalkyl. In some cases, the alkylene group isa lower alkylene group (i.e., a C₁₋₄ alkylene group).

As used herein, “heteroaryl” refers to an aromatic ring or ring system(i.e., two or more fused rings that share two adjacent atoms) thatcontain(s) one or more heteroatoms, that is, an element other thancarbon, including but not limited to, nitrogen, oxygen and sulfur, inthe ring backbone. When the heteroaryl is a ring system, every ring inthe system is aromatic. The heteroaryl group may have 5-18 ring members(i.e., the number of atoms making up the ring backbone, including carbonatoms and heteroatoms), although the present definition also covers theoccurrence of the term “heteroaryl” where no numerical range isdesignated. In some embodiments, the heteroaryl group has 5 to 10 ringmembers or 5 to 7 ring members. The heteroaryl group may be designatedas “5-7 membered heteroaryl,” “5-10 membered heteroaryl,” or similardesignations. Examples of heteroaryl rings include, but are not limitedto, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl,imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl,thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,quinolinyl, isoquinlinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl,indolyl, isoindolyl, and benzothienyl.

A “heteroaralkyl” or “heteroarylalkyl” is heteroaryl group connected, asa substituent, via an alkylene group. Examples include but are notlimited to 2-thienylmethyl, 3-thienylmethyl, furylmethyl, thienylethyl,pyrrolylalkyl, pyridylalkyl, isoxazollylalkyl, and imidazolylalkyl. Insome cases, the alkylene group is a lower alkylene group (i.e., a C₁₋₄alkylene group).

As used herein, “carbocyclyl” means a non-aromatic cyclic ring or ringsystem containing only carbon atoms in the ring system backbone. Whenthe carbocyclyl is a ring system, two or more rings may be joinedtogether in a fused, bridged or spiro-connected fashion. Carbocyclylsmay have any degree of saturation provided that at least one ring in aring system is not aromatic. Thus, carbocyclyls include cycloalkyls,cycloalkenyls, and cycloalkynyls. The carbocyclyl group may have 3 to 20carbon atoms, although the present definition also covers the occurrenceof the term “carbocyclyl” where no numerical range is designated. Thecarbocyclyl group may also be a medium size carbocyclyl having 3 to 10carbon atoms. The carbocyclyl group could also be a carbocyclyl having 3to 6 carbon atoms. The carbocyclyl group may be designated as “C₃₋₆carbocyclyl” or similar designations. Examples of carbocyclyl ringsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicycle[2.2.2]octanyl,adamantyl, and spiro[4.4]nonanyl.

A “(carbocyclyl)alkyl” is a carbocyclyl group connected, as asubstituent, via an alkylene group, such as “C₄₋₁₀ (carbocyclyl)alkyl”and the like, including but not limited to, cyclopropylmethyl,cyclobutylmethyl, cyclopropylethyl, cyclopropylbutyl, cyclobutylethyl,cyclopropylisopropyl, cyclopentylmethyl, cyclopentylethyl,cyclohexylmethyl, cyclohexylethyl, cycloheptylmethyl, and the like. Insome cases, the alkylene group is a lower alkylene group.

As used herein, “cycloalkyl” means a fully saturated carbocyclyl ring orring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl.

As used herein, “cycloalkenyl” means a carbocyclyl ring or ring systemhaving at least one double bond, wherein no ring in the ring system isaromatic. An example is cyclohexenyl.

As used herein, “heterocyclyl” means a non-aromatic cyclic ring or ringsystem containing at least one heteroatom in the ring backbone.Heterocyclyls may be joined together in a fused, bridged orspiro-connected fashion. Heterocyclyls may have any degree of saturationprovided that at least one ring in the ring system is not aromatic. Theheteroatom(s) may be present in either a non-aromatic or aromatic ringin the ring system. The heterocyclyl group may have 3 to 20 ring members(i.e., the number of atoms making up the ring backbone, including carbonatoms and heteroatoms), although the present definition also covers theoccurrence of the term “heterocyclyl” where no numerical range isdesignated. The heterocyclyl group may also be a medium sizeheterocyclyl having 3 to 10 ring members. The heterocyclyl group couldalso be a heterocyclyl having 3 to 6 ring members. The heterocyclylgroup may be designated as “3-6 membered heterocyclyl” or similardesignations. In preferred six membered monocyclic heterocyclyls, theheteroatom(s) are selected from one up to three of O (oxygen), N(nitrogen) or S (sulfur), and in preferred five membered monocyclicheterocyclyls, the heteroatom(s) are selected from one or twoheteroatoms selected from O (oxygen), N (nitrogen) or S (sulfur).Examples of heterocyclyl rings include, but are not limited to,azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl,imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl,piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl,pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl,1,3-dioxanyl, 1,4-dioxinyl, 1,4-dioxanyl, 1,3-oxathianyl,1,4-oxathiinyl, 1,4-oxathianyl, 2H-1,2-oxazinyl, trioxanyl,hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithiolyl,1,3-dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl,oxazolidinonyl, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolinyl,isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl,thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, andtetrahydroquinoline.

A “(heterocyclyl)alkyl” is a heterocyclyl group connected, as asubstituent, via an alkylene group. Examples include, but are notlimited to, imidazolinylmethyl and indolinylethyl.

As used herein, “acyl” refers to —C(═O)R, wherein R is hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.Non-limiting examples include formyl, acetyl, propanoyl, benzoyl, andacryl.

As used herein, “alkanoyl” refers to a “carbonyl” substituted with an“alkyl” group, the “alkanoyl” group is covalently bonded to the parentmolecule through the carbon of the “carbonyl” group. Non-limitingexamples include methanoyl, ethanoyl, and propanoyl.

An “O-carboxy” group refers to a “—OC(═O)R” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein.

A “C-carboxy” group refers to a “—C(═O)OR” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein. A non-limiting example includes carboxyl (i.e.,—C(═O)OH).

A “cyano” group refers to a “—CN” group.

A “cyanato” group refers to an “—OCN” group.

An “isocyanato” group refers to a “—NCO” group.

A “thiocyanato” group refers to a “—SCN” group.

An “isothiocyanato” group refers to an “—NCS” group.

A “sulfinyl” group refers to an “—S(═O)R” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein.

A “sulfonyl” group refers to an “—SO₂R” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein.

An “S-sulfonamido” group refers to a “—SO₂NR_(A)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “N-sulfonamido” group refers to a “—N(R_(A))SO₂R_(B)” group in whichR_(A) and R_(b) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “O-carbamyl” group refers to a “—OC(═O)NR_(A)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “N-carbamyl” group refers to an “—N(R_(A))C(═O)OR_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “O-thiocarbamyl” group refers to a “—OC(═S)NR^(A)R^(B)” group inwhich R_(A) and R_(B) are each independently selected from hydrogen,C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl,5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as definedherein.

A “urea” group refers to a “—N(R_(A))C(═O)NR_(A)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “N-thiocarbamyl” group refers to an “—N(R_(A))C(═S)OR_(B)” group inwhich R_(A) and R_(B) are each independently selected from hydrogen,C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl,5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as definedherein.

A “C-amido” group refers to a “—C(═O)NR_(A)R_(B)” group in which R_(A)and R_(B) are each independently selected from hydrogen, C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “N-amido” group refers to a “—N(R_(A))C(═O)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “amino” group refers to a “—NR_(A)R_(B)” group in which R_(A) andR_(B) are each independently selected from hydrogen, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, and 5-10 membered heterocyclyl, as defined herein. Anon-limiting example includes free amino (i.e., —NH₂).

An “aminoalkyl” group refers to an amino group connected via an alkylenegroup.

An “alkoxyalkyl” group refers to an alkoxy group connected via analkylene group, such as a “C₂₋₈ alkoxyalkyl” and the like.

As used herein, a substituted group is derived from the unsubstitutedparent group in which there has been an exchange of one or more hydrogenatoms for another atom or group. Unless otherwise indicated, when agroup is deemed to be “substituted,” it is meant that the group issubstituted with one or more substitutents independently selected fromC₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇ carbocyclyl (optionallysubstituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, andC₁-C₆ haloalkoxy), C₃-C₇-carbocyclyl-C₁-C₆-alkyl (optionally substitutedwith halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆haloalkoxy), 5-10 membered heterocyclyl (optionally substituted withhalo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),5-10 membered heterocyclyl-C₁-C₆-alkyl (optionally substituted withhalo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),aryl (optionally substituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆haloalkyl, and C₁-C₆ haloalkoxy), aryl(C₁-C₆)alkyl (optionallysubstituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, andC₁-C₆ haloalkoxy), 5-10 membered heteroaryl (optionally substituted withhalo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),5-10 membered heteroaryl(C₁-C₆)alkyl (optionally substituted with halo,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), halo,cyano, hydroxy, C₁-C₆ alkoxy, C₁-C₆ alkoxy(C₁-C₆)alkyl (i.e., ether),aryloxy, sulfhydryl (mercapto), halo(C₁-C₆)alkyl (e.g., —CF₃),halo(C₁-C₆)alkoxy (e.g., —OCF₃), C₁-C₆ alkylthio, arylthio, amino,amino(C₁-C₆)alkyl, nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido,C-carboxy, O-carboxy, acyl, cyanato, isocyanato, thiocyanato,isothiocyanato, sulfinyl, sulfonyl, and oxo (═O). Wherever a group isdescribed as “optionally substituted” that group can be substituted withthe above substituents.

It is to be understood that certain radical naming conventions caninclude either a mono-radical or a di-radical, depending on the context.For example, where a substituent requires two points of attachment tothe rest of the molecule, it is understood that the substituent is adi-radical. For example, a substituent identified as alkyl that requirestwo points of attachment includes di-radicals such as —CH₂—, —CH₂CH₂—,—CH₂CH(CH₃)CH₂—, and the like. Other radical naming conventions clearlyindicate that the radical is a di-radical such as “alkylene” or“alkenylene.”

When two R groups are said to form a ring (e.g., a carbocyclyl,heterocyclyl, aryl, or heteroaryl ring) “together with the atom to whichthey are attached,” it is meant that the collective unit of the atom andthe two R groups are the recited ring. The ring is not otherwise limitedby the definition of each R group when taken individually. For example,when the following substructure is present:

-   -   and R¹ and R² are defined as selected from the group consisting        of hydrogen and alkyl, or R¹ and R² together with the nitrogen        to which they are attached form a heterocyclyl, it is meant that        R¹ and R² can be selected from hydrogen or alkyl, or        alternatively, the substructure has structure:

-   -   where ring E is a heteroaryl ring containing the depicted        nitrogen.

Similarly, when two “adjacent” R groups are said to form a ring“together with the atom to which they are attached,” it is meant thatthe collective unit of the atoms, intervening bonds, and the two Rgroups are the recited ring. For example, when the followingsubstructure is present:

-   -   and R¹ and R² are defined as selected from the group consisting        of hydrogen and alkyl, or R¹ and R² together with the atoms to        which they are attached form an aryl or carbocylyl, it is meant        that R¹ and R² can be selected from hydrogen or alkyl, or        alternatively, the substructure has structure:

-   -   where E is an aryl ring or a carbocylyl containing the depicted        double bond.

Wherever a substituent is depicted as a di-radical (i.e., has two pointsof attachment to the rest of the molecule), it is to be understood thatthe substituent can be attached in any directional configuration unlessotherwise indicated. Thus, for example, a substituent depicted as -AE-or

includes the substituent being oriented such that the A is attached atthe leftmost attachment point of the molecule as well as the case inwhich A is attached at the rightmost attachment point of the molecule.

“Subject” as used herein, means a human or a non-human mammal, e.g., adog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-humanprimate or a bird, e.g., a chicken, as well as any other vertebrate orinvertebrate.

The term “antibody-drug conjugate” refers to molecular entity thatincludes an antibody linked to a drug moiety, optionally via a chemicallinker.

Compounds

The skilled artisan will recognize that some structures described hereinmay be resonance forms or tautomers of compounds that may be fairlyrepresented by other chemical structures, even when kinetically; theartisan recognizes that such structures may only represent a very smallportion of a sample of such compound(s). Such compounds are consideredwithin the scope of the structures depicted, though such resonance formsor tautomers are not represented herein.

Isotopes may be present in the compounds described. Each chemicalelement present in a compound either specifically or genericallydescribed hereinmay include any isotope of said element. For example, ina compound specifically or generically described herein a hydrogen atommay be explicitly disclosed or understood to be present in the compoundand each such hydrogen atom is any isotope of hydrogen, including butnot limited to hydrogen-1 (protium) and hydrogen-2 (deuterium). Thus,reference herein to a compound encompasses all potential isotopic formsunless the context clearly dictates otherwise.

Utilities and Applications

Some embodiments provide a method of treating a patient in need thereofcomprising administering a compound as disclosed and described herein tosaid patient. In some embodiments, the patient may have cancer. In someembodiments, the compound may have anti-tumor activity.

Structures

Some embodiments provide a compound having the structure of Formula I:

-   -   or pharmaceutically acceptable salts or solvents thereof,        wherein:    -   R¹ is selected from the group consisting of hydrogen, deuterium,        an optionally substituted C₁-C₆ alkyl, an optionally substituted        C₃-C₇ cycloalkyl, an optionally substituted aryl, and an        optionally substituted heteroaryl; R² is selected from the group        consisting of hydrogen, deuterium, an optionally substituted        C₁-C₆ alkyl, an optionally substituted C₃-C₇ cycloalkyl, an        optionally substituted aryl, and an optionally substituted        heteroaryl;    -   R³ is selected from the group consisting of hydrogen, deuterium,        and optionally substituted C₁-C₆ alkyl, an optionally        substituted hydroxyl, an optionally substituted C₁-C₆ alkoxy, an        optionally substituted C₃-C₇ cycloalkyl, an optionally        substituted aryl, and an optionally substituted heteroaryl;    -   R⁴ is an optionally substituted C₁-C₆ alkyl;    -   R⁵ is hydrogen, deuterium or an optionally substituted C₁-C₆        alkyl;    -   R⁶ is hydrogen, deuterium or an optionally substituted C₁-C₆        alkyl;    -   X¹ and X² are each independently selected from the group        consisting of hydrogen, deuterium, an optionally substituted        C₁-C₆ alkyl, an optionally substituted aryl, an optionally        substituted heteroaryl, halogen, —CN, —N₃, —COOR^(B),        —NR^(A)R^(B), —OR^(B), and —SR^(B), where at least one of X¹ and        X² is selected from the group consisting of an optionally        substituted aryl, an optionally substituted heteroaryl, halogen,        —CN, —N₃, —COOR^(B), —NR^(A)R^(B), OR^(B), and —SR^(B) when R⁵        is methyl;    -   R^(A) is selected from the group consisting of hydrogen,        deuterium, an optionally substituted C₁-C₆ alkyl, an optionally        substituted C₃-C₇ cycloalkyl, an optionally substituted aryl,        and an optionally substituted heteroaryl;    -   R^(B) is selected from the group consisting of hydrogen,        deuterium, an optionally substituted C₁-C₆ alkyl, an optionally        substituted C₃-C₇ cycloalkyl, an optionally substituted aryl,        and an optionally substituted heteroaryl;    -   R⁷ is an optionally substituted C₁-C₆ alkyl, an optionally        substituted heteroaryl, or —C(═O)R^(C);    -   R^(C) is selected from the group consisting of an optionally        substituted C₁-C₆ alkyl, optionally substituted C₁-C₆ alkoxy, an        optionally substituted C₃-C₇ cycloalkyl, an optionally        substituted aryl, and an optionally substituted heteroaryl, and        hydroxyl; and    -   R⁸ is hydrogen, deuterium or hydroxyl

In some embodiments, the compound having the structure of Formula I hasthe structure Formula Ia:

-   -   or pharmaceutically acceptable salts or solvates thereof.

In some embodiments, the compound having the structure of Formula I hasthe structure Formula Ib:

-   -   or pharmaceutically acceptable salts or solvates thereof.

In some embodiments, the compound having the structure of Formula I hasthe structure Formula Ic:

-   -   or pharmaceutically acceptable salts or solvates thereof.

Some embodiments provide a compound having the structure of Formula II:

-   -   or pharmaceutically acceptable salts or solvates thereof,    -   wherein:    -   R¹ is selected from the group consisting of hydrogen, deuterium,        an optionally substituted C₁-C₆ alkyl, an optionally substituted        C₃-C₇ cycloalkyl, an optionally substituted aryl, and an        optionally substituted heteroaryl;    -   R² is selected from the group consisting of R^(D), an optionally        substituted C₁-C₆ alkyl, an optionally substituted C₃-C₇        cycloalkyl, an optionally substituted aryl, and an optionally        substituted heteroaryl;    -   R³ is selected from the group consisting of hydrogen, deuterium,        and optionally substituted C₁-C₆ alkyl, an optionally        substituted hydroxyl, an optionally substituted C₁-C₆ alkoxy, an        optionally substituted C₃-C₇ cycloalkyl, an optionally        substituted aryl, and an optionally substituted heteroaryl;    -   R⁴ is an optionally substituted C₁-C₆ alkyl;    -   R⁵ is hydrogen, deuterium or an optionally substituted C₁-C₆        alkyl;    -   R⁶ is hydrogen, deuterium or an optionally substituted C₁-C₆        alkyl;    -   X¹ and X² are each independently selected from the group        consisting of hydrogen, deuterium, an optionally substituted        C₁-C₆ alkyl, an optionally substituted aryl, an optionally        substituted heteroaryl, halogen, —CN, —N₃, —COOR^(B),        —NR^(A)R^(B), —OR^(B), and —SR^(B), where at least one of X¹ and        X² is selected from the group consisting of an optionally        substituted aryl, an optionally substituted heteroaryl, halogen,        —CN, —N₃, —COOR^(B), —NR^(A)R^(B), OR^(B), and —SR^(B) when R⁵        is methyl;    -   R^(A) is selected from the group consisting of hydrogen,        deuterium, an optionally substituted C₁-C₆ alkyl, an optionally        substituted C₃-C₇ cycloalkyl, an optionally substituted aryl,        and an optionally substituted heteroaryl;    -   R^(B) is selected from the group consisting of hydrogen,        R^(1A)-L^(I), R^(3A)-L¹-, R^(4A)-L¹-, Mc-L¹-, Mal-L³-L¹-,        R^(1A)-Mal-L³-L¹-Val-Cit-PAB—C(O)— or        Mal-L³-L¹-Val-Cit-PAB—C(O)—;    -   R⁷ is an optionally substituted C₁-C₆ alkyl, an optionally        substituted heteroaryl, or —C(═O)R^(C);    -   R^(C) is selected from the group consisting of an optionally        substituted C₁-C₆ alkyl, optionally substituted C₁-C₆ alkoxy, an        optionally substituted C₃-C₇ cycloalkyl, an optionally        substituted aryl, and an optionally substituted heteroaryl, and        hydroxyl; and    -   R⁸ is hydrogen, deuterium, —OR^(D), or —SR^(D);    -   R^(D) is selected from the group consisting of hydrogen,        R^(1A)-L, R^(3A)-L¹-, R^(4A)-L¹-, Mc-L¹-, Mal-L³-L¹-,        R^(1A)-Mal-L³-L¹-Val-Cit-PAB—C(O)— and        Mal-L³-L¹-Val-Cit-PAB—C(O)—;    -   R^(3A) is

-   -   R^(4A) is a conjugation moiety;    -   R^(1A) is a conjugated targeting moiety;    -   L¹ is a linker or a bond; and    -   L³ is an alkanoyl.

In some embodiments, the compound having the structure of Formula II hasthe structure Formula IIa:

-   -   or pharmaceutically acceptable salts or solvates thereof,    -   wherein R^(D) is selected from the group consisting of R^(1A)-L,        R^(3A)-L¹-, R^(4A)-L¹-, Mc-L¹-, Mal-L³-L¹-,        R^(1A)-Mal-L³-L¹-Val-Cit-PAB—C(O)— and        Mal-L³-L¹-Val-Cit-PAB—C(O)—.

In some embodiments, the compound having the structure of Formula II hasthe structure Formula IIb:

-   -   or pharmaceutically acceptable salts or solvates thereof,    -   wherein R^(B) is selected from the group consisting of R^(1A)-L,        R^(3A)-L¹-, R^(4A)-L¹-, Mc-L¹-, Mal-L³-L¹-,        R^(1A)-Mal-L³-L¹-Val-Cit-PAB—C(O)— and        Mal-L³-L¹-Val-Cit-PAB—C(O)—.

In some embodiments, the compound having the structure of Formula II hasthe structure Formula IIc:

-   -   or pharmaceutically acceptable salts or solvates thereof,    -   wherein R^(B) is selected from the group consisting of        R^(1A)-L¹-, R^(3A)-L¹-, R^(4A)-L¹-, Mc-L¹-, Mal-L³-L¹-,        R^(1A)-Mal-L³-L¹-Val-Cit-PAB—C(O)— and        Mal-L³-L¹-Val-Cit-PAB—C(O)—.

In some embodiments, the compound having the structure of Formula II hasthe structure Formula IId:

-   -   or pharmaceutically acceptable salts or solvates thereof,    -   wherein R^(D) is selected from the group consisting of        R^(1A)-L¹-, R^(3A)-L¹-, R^(4A)-L¹-, Mc-L¹-, Mal-L³-L¹-,        R^(1A)-Mal-L³-L¹-Val-Cit-PAB—C(O)— and        Mal-L³-L¹-Val-Cit-PAB—C(O)—.

In some embodiments of a compound having the structure of Formula II,IIa and IId, R^(D) is R^(1A)-Mc-Val-Cit-PAB—C(O)— orR^(1A)-Mal-L³-Val-Cit-PAB-C(O)—. In some embodiments of a compoundhaving the structure of Formula II, IIb and IIc, R^(B) isR^(1A)-Mc-Val-Cit-PAB—C(O)— or R^(1A)-Mal-L³-Val-Cit-PAB—C(O)—. In someembodiments of a compound having the structure of Formula II, IIa andIId, R^(D) is Mc-Val-Cit-PAB—C(O)— or Mal-L³-Val-Cit-PAB—C(O)—. In someembodiments of a compound having the structure of Formula II, IIb andIIc, R^(B) is Mc-Val-Cit-PAB—C(O)— or Mal-L³-Val-Cit-PAB—C(O)—.

In some embodiments of a compound having the structure of Formula II,IIa and IId, R^(D) may be

In some embodiments of a compound having the structure of Formula II,IIb and IIc, R^(B) may be

In some embodiments of a compound having the structure of Formula II,IIa and IId, R^(D) may be

In some embodiments of a compound having the structure of Formula II,IIb and IIc, R^(B) may be

In some embodiments, the targeting moiety binds to one or moretumor-associated antigens or cell surface receptors selected from thegroup consisting of CD19, CD22, CD30, CD33, CD56, CD70, CD79b, CD74,CD138, HER2, GPNMB, PSMA, SLC44A4, CA6, CA-IX, Mesothelin, CD66e,CEACAM5, and Nectin-4.

In some embodiments, the R¹ comprises a targeting moiety selected fromthe group consisting of brentuximab, inotuzumab, gemtuzumab,milatuzumab, trastuzumab, glembatumomab, lorvotuzumab, or labestuzumab,or derivatives thereof.

In some embodiments, the targeting moiety is a monoclonal antibody(mAB). In some embodiments, the targeting moiety is an antibodyfragment, surrogate, or variant. In some embodiments, the targetingmoiety is a protein ligand. In some embodiments, the targeting moiety isa protein scaffold. In some embodiments, the targeting moiety is apeptide, cysteine-engineered antibody or antibody-like protein. In someembodiments, the targeting moiety is a small molecule ligand or nucleicacid aptamer.

In some embodiments, L¹ may be—(CHR¹³)—CH₂—(CR¹⁴R¹⁵)—S—S—(CR¹⁶R¹⁷)—(CH₂)_(n)(CO)_(r)—; n is 1, 2, 3,4, or 5; r is 0 or 1; R¹³ is hydrogen or SO₃H; and R¹⁴, R¹⁵, R¹⁶ and R¹⁷are each independently hydrogen or an optionally substituted C₁-C₆alkyl. In some embodiments, L¹ comprises.

In some embodiments, L¹ comprises a dipeptide selected from the groupconsisting of -Phe-Lys-, -Val-Ala-, -Val-Lys-; -Ala-Lys-, -Val-Cit-,-Phe-Cit-, -Leu-Cit-, -Ile-Cit-, -Phe-Arg-, and -Trp-Cit-. In someembodiments, R^(1A)-L¹- is

q is 0 to 6; and L⁴ is a linker. In some embodiments, R^(1A)-L¹- is

s is 0 or 1; and t is 0 to 30. In some embodiments, L¹- comprises

In some embodiments, R^(1A)-L¹- is

where t is 0 to 30. In some embodiments, R^(4A)— is

and

-   -   R is hydrogen, —C(═O)N(CH₂CH₃)₂, or —SO₂N(CH₂CH₂)₂O.

In some embodiments, R^(4A)— is

In some embodiments, L¹ is L^(1A)-L^(2A); L^(1A) comprises at least onemoiety selected from the group consisting of

-   -   L^(2A) comprises at least one moiety selected from the group        consisting of

-   -   n is 0 or 1; and R is hydrogen or alkyl.

In some embodiments of a compound having the structure of Formula II, R¹is R^(D).

In some embodiments, the compound having the structure of Formula I isnot

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound having the structure of Formula I is

or a pharmaceutically acceptable salt thereof.

Also provided herein are the compounds described above conjugated to atargeting moiety with a linker. Also provided herein are the compoundsdescribed above with a linker.

In some embodiments, the compound is conjugated to a targeting moiety.

In some embodiments, the targeting moieties can be an antibody, antibodyfragment, antibody-like protein or nucleic acid aptamer.

In some embodiments, the targeting moiety includes a monoclonal antibody(mAB). In some embodiments, the compound includes a spacer or a linker.

Conjugation Methods, Spacers and Linkers Involved

Some embodiments provide a method of conjugating of a targeting moietythrough a linker.

In some embodiments, the method includes a single-step or sequentialconjugation approach. In some embodiments, the compound includes alinker. In some embodiments, the linker may include a noncleavable orcleavable unit such as peptides.

In some embodiments of a compound represented by Formula II, R^(1A)-L¹-has a structure selected from:

wherein A is the targeting moiety. In some embodiments, L¹ may be—(CH₂O)_(n) (where n is 1, 2, 3, 4, 5, 6, or 7), optionally substitutedC₁-C₈ alkyl, optionally substituted C₃-C₈ cycloalkyl, optionallysubstituted C₁-C₈ alkoxy, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted heterocyclyl, orcombination thereof. In some embodiments, L¹ is an optionallysubstituted C₁-C₈ alkyl. In some embodiments, L⁵ is C₁-C₈ alkyl.

In some embodiments of a compound represented by Formula II, R^(1A)-L¹-has a structure selected from:

wherein A is the targeting moiety. In some embodiments, L¹ may be—(CH₂O)_(n) (where n is 1, 2, 3, 4, 5, 6, or 7), optionally substitutedC₁-C₈ alkyl, optionally substituted C₃-C₈ cycloalkyl, optionallysubstituted C₁-C₈ alkoxy, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted heterocyclyl, orcombination thereof. In some embodiments, L¹ is an optionallysubstituted C₁-C₈ alkyl. In some embodiments, L⁵ is C₁-C₈ alkyl.

In some embodiments, the targeting moiety may be a monoclonal antibody(mAB). In some embodiments, the targeting moiety may be an antibodyfragment, surrogate, or variant. In some embodiments, the targetingmoiety may be a protein ligand. In some embodiments, the targetingmoiety may be a protein scaffold. In some embodiments, the targetingmoiety may be a peptide, cysteine-engineered antibody or antibody-likeprotein. In some embodiments, the targeting moiety may be RNA or DNA. Insome embodiments, the targeting moiety may be a RNA or DNA fragment.

In some embodiments, L¹ may be a peptide. In some embodiments, L₁ mayinclude —(CH₂)_(n)— where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In someembodiments, L¹ may include —(CH₂CH₂O)_(n)— where n is 1, 2, 3, 4, 5, 6,7, 8, 9, or 10. In some embodiments, L¹ may include Val-Ala-, Phe-Lys-,D-Val-Leu-Lay, Gly-Gly-Arg, Ala-Ala-Asn-, or the like. In someembodiments, L¹ may include any combination of peptide, oligosaccharide,—(CH₂)_(n)—, —(CH₂CH₂O)_(n)—, Val-Ala-, Phe-Lys-, D-Val-Leu-Lay,Gly-Gly-Arg, Ala-Ala-Asn-, and the like. In some embodiments, the L¹ mayinclude a peptide. In some embodiments, L¹ may be—(CHR¹³)—CH₂—(CR¹⁴R¹⁵)—S—S—(CR¹⁶R¹⁷)—(CH2)_(n)(CO)_(r)—; n may be 1, 2,3, 4, or 5; r may be 0 or 1; R¹³ may be hydrogen or SO₃H; and R¹⁴, Ris,R and R¹⁷ may each be independently hydrogen or an optionallysubstituted C₁-C₆ alkyl.

In some embodiments, L¹ may comprise

In some embodiments, the compound-conjugates may include one or morecomponents selected from the group consisting of an amino acid, an aminoacid residue, an amino acid analog, and a modified amino acid. In someembodiments of a compound represented by Formula II, L¹ comprises adipeptide selected from the group consisting of -Phe-Lys-, -Val-Ala-,-Val-Lys-; -Ala-Lys-, -Val-Cit-, -Phe-Cit-, -Leu-Cit-, -Ile-Cit-,-Phe-Arg-, and -Trp-Cit-.

In some embodiments of a compound represented by Formula II, L¹comprises at least one selected from the group consisting of: anoptionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, an optionally substituted alkoxy, an optionallysubstituted cycloalkyl, an optionally substituted aryl, optionallysubstituted heterocycyl, and an optionally substituted heteroaryl.

In some embodiments of a compound represented by Formula II, L¹comprises at least one selected from the group consisting of: adisulfide, an ester, a carbamate, a ketal, a urea and a urethane.

In some embodiments of a compound represented by Formula II, R^(1A)-L¹-is

q is 0 to 6; and L⁴ is a linker.

In some embodiments of a compound represented by Formula II, R^(1A)-L¹-is

s is 0 or 1; and t is 0 to 30.

In some embodiments of a compound represented by Formula II, R^(1A)-L¹-is

-   -   where t is 0 to 30. In some embodiments, R^(1A) may be an        antibody fragment, surrogate, or variant. In some embodiments,        R^(1A) may be a monoclonal antibody (mAB). In some embodiments,        R^(1A) may be a targeting moiety selected from the group        consisting of brentuximab, inotuzumab, gemtuzumab, milatuzumab,        trastuzumab, glembatumomab, lorvotuzumab, or labestuzumab, or        derivatives thereof.

As used herein, the term “peptide” refers to a structure including oneor more components each individually selected from the group consistingof an amino acid, an amino acid residue, an amino acid analog, and amodified amino acid. The components are typically joined to each otherthrough an amide bond.

As used herein, the term “amino acid” includes naturally occurring aminoacids, a molecule having a nitrogen available for forming an amide bondand a carboxylic acid, a molecule of the general formula NH₂—CHR—COOH orthe residue within a peptide bearing the parent amino acid, where “R” isone of a number of different side chains. “R” can be a substituent foundin naturally occurring amino acids. “R” can also be a substituentreferring to one that is not of the naturally occurring amino acids.

As used herein, the term “amino acid residue” refers to the portion ofthe amino acid which remains after losing a water molecule when it isjoined to another amino acid.

As used herein, the term “amino acid analog” refers to a structuralderivative of an amino acid parent compound that often differs from itby a single element.

As used herein, the term “modified amino acid” refers to an amino acidbearing an “R” substituent that does not correspond to one of the twentygenetically coded amino acids.

As used herein, the abbreviations for the genetically encodedL¹-enantiomeric amino acids are conventional and are as follows: TheD-amino acids are designated by lower case, e.g. D-proline=p, etc.

TABLE 1 Amino Acids One-Letter Symbol Common Abbreviation Alanine A AlaArginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C CysGlutamine Q Gln Glutamic acid E Glu Glycine G Gly Histidine H HisIsoleucine I Ile Leucine L Leu Lysine K Lys Phenylalanine F Phe ProlineP Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y TyrValine V Val

Certain amino acid residues in the compound-conjugate can be replacedwith other amino acid residues without significantly deleteriouslyaffecting, and in many cases even enhancing, the activity of thepeptides. Thus, also contemplated by the preferred embodiments arealtered or mutated forms of the active agent-conjugate wherein at leastone defined amino acid residue in the structure is substituted withanother amino acid residue or derivative and/or analog thereof. It willbe recognized that in preferred embodiments, the amino acidsubstitutions are conservative, i.e., the replacing amino acid residuehas physical and chemical properties that are similar to the amino acidresidue being replaced.

For purposes of determining conservative amino acid substitutions, theamino acids can be conveniently classified into two maincategories—hydrophilic and hydrophobic—depending primarily on thephysical-chemical characteristics of the amino acid side chain. Thesetwo main categories can be further classified into subcategories thatmore distinctly define the characteristics of the amino acid sidechains. For example, the class of hydrophilic amino acids can be furthersubdivided into acidic, basic and polar amino acids. The class ofhydrophobic amino acids can be further subdivided into nonpolar andaromatic amino acids. The definitions of the various categories of aminoacids are as follows:

The term “hydrophilic amino acid” refers to an amino acid exhibiting ahydrophobicity of less than zero according to the normalized consensushydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol.179:125-142. Genetically encoded hydrophilic amino acids include Thr(T), Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K) andArg (R).

The term “hydrophobic amino acid” refers to an amino acid exhibiting ahydrophobicity of greater than zero according to the normalizedconsensus hydrophobicity scale of Eisenberg, 1984, J. Mol. Biol.179:1.25-142. Genetically encoded hydrophobic amino acids include Pro(P), Ile (I), Phe (F), Val (V), Leu (L), Trp (W), Met (M), Ala (A), Gly(G) and Tyr (Y).

The term “acidic amino acid” refers to a hydrophilic amino acid having aside chain pK value of less than 7. Acidic amino acids typically havenegatively charged side chains at physiological pH due to loss of ahydrogen ion. Genetically encoded acidic amino acids include Glu (E) andAsp (D).

The term “basic amino acid” refers to a hydrophilic amino acid having aside chain pK value of greater than 7. Basic amino acids typically havepositively charged side chains at physiological pH due to associationwith hydronium ion. Genetically encoded basic amino acids include His(H), Arg (R) and Lys (K).

The term “polar amino acid” refers to a hydrophilic amino acid having aside chain that is uncharged at physiological pH, but which has at leastone bond in which the pair of electrons shared in common by two atoms isheld more closely by one of the atoms. Genetically encoded polar aminoacids include Asn (N), Gln (Q) Ser (S) and Thr (T).

The term “nonpolar amino acid” refers to a hydrophobic amino acid havinga side chain that is uncharged at physiological pH and which has bondsin which the pair of electrons shared in common by two atoms isgenerally held equally by each of the two atoms (i.e., the side chain isnot polar). Genetically encoded nonpolar amino acids include Leu (L),Val (V), Ile (I), Met (M), Gly (G) and Ala (A).

The term “aromatic amino acid” refers to a hydrophobic amino acid with aside chain having at least one aromatic or heteroaromatic ring. In someembodiments, the aromatic or heteroaromatic ring may contain one or moresubstituents such as —OH, —SH, —CN, —F, —Cl, —Br, —I, —NO₂, —NO, —NH₂,—NHR, —NRR, —C(O)R, —C(O)OH, —C(O)OR, —C(O)NH₂, —C(O)NHR, —C(O)NRR andthe like where each R is independently (C₁-C₆) alkyl, substituted(C₁-C₆) alkyl, (C₁-C₆) alkenyl, substituted (C₁-C₆) alkenyl, (C₁-C₆)alkynyl, substituted (C₁-C₆) alkynyl, (C₅-C₂₀) aryl, substituted(C₅-C₂₀) aryl, (C₆-C₂₆) alkaryl, substituted (C₆-C₂₆) alkaryl, 5-20membered heteroaryl, substituted 5-20 membered heteroaryl, 6-26 memberedalkheteroaryl or substituted 6-26 membered alkheteroaryl. Geneticallyencoded aromatic amino acids include Phe (F), Tyr (Y) and Trp (W).

The term “aliphatic amino acid” refers to a hydrophobic amino acidhaving an aliphatic hydrocarbon side chain. Genetically encodedaliphatic amino acids include Ala (A), Val (V), Leu (L) and Ile (I).

The amino acid residue Cys (C) is unusual in that it can form disulfidebridges with other Cys (C) residues or other sulfanyl-containing aminoacids. The ability of Cys (C) residues (and other amino acids with —SHcontaining side chains) to exist in a peptide in either the reduced free—SH or oxidized disulfide-bridged form affects whether Cys (C) residuescontribute net hydrophobic or hydrophilic character to a peptide. WhileCys (C) exhibits a hydrophobicity of 0.29 according to the normalizedconsensus scale of Eisenberg (Eisenberg, 1984, supra), it is to beunderstood that for purposes of the preferred embodiments Cys (C) iscategorized as a polar hydrophilic amino acid, notwithstanding thegeneral classifications defined above.

As used herein, the term “conjugation moiety” refers to a molecularentity that includes a group that forms a covalent bond by reacting witha function group of a targeting moiety. In some embodiments, theconjugation moiety may react with an amino acid functional group of amonoclonal antibody (mAB).

As used herein, the term “targeting moiety” refers to a structure thatbinds or associates with a biological moiety or fragment thereof.

In some embodiments, the targeting moiety may be a monoclonal antibody(mAB). In some embodiments, the targeting moiety may be an antibodyfragment, surrogate, or variant. In some embodiments, the targetingmoiety may be a protein ligand. In some embodiments, the targetingmoiety may be a protein scaffold. In some embodiments, the targetingmoiety may be a peptide. In some embodiments, the targeting moiety maybe RNA or DNA. In some embodiments, the targeting moiety may be a RNA orDNA fragment. In some embodiments, the targeting moiety may be a smallmolecule ligand or nucleic acid aptamer.

In some embodiments, the targeting moiety may be an antibody fragmentdescribed in Janthur et al., “Drug Conjugates Such as Antibody DrugConjugates (ADCs), Immunotoxins and Immunoliposomes Challenge DailyClinical Practice,” Int. J. Mol. Sci. 2012, 13, 16020-16045, thedisclosure of which is incorporated herein by reference in its entirety.In some embodiments, the targeting moiety may be an antibody fragmentdescribed in Trail, P A, “Antibody Drug Conjugates as CancerTherapeutics,” Antibodies 2013, 2, 113-129, the disclosure of which isincorporated herein by reference in its entirety.

In some embodiments, the targeting moiety may be an anti-HER2 antibody.In some embodiments, the targeting moiety may be an anti-EGFR antibody.

In some embodiments, the targeting moiety may be HuM195-Ac-225,HuM195-Bi-213, Anyara (naptumomab estafenatox; ABR-217620), AS1409,Zevalin (ibritumomab tiuxetan), BIIB015, BT-062, Neuradiab, CDX-1307,CR011-vcMMAE, Trastuzumab-DM1 (R3502), Bexxar (tositumomab), IMGN242,IMGN388, IMGN901, ¹³¹I-labetuzumab, IMMU-102 (⁹⁰Y-epratuzumab), IMMU-107(⁹⁰Y-clivatuzumab tetraxetan), MDX-1203, CAT-8015, EMD 273063(hu14.18-IL2), Tucotuzumab celmoleukin (EMD 273066; huKS-IL2),¹⁸⁸Re-PTI-6D2, Cotara, L¹⁹-IL2, Teleukin (F16-IL2), Tenarad (F16-¹³¹I),L¹⁹-¹³¹I, L¹⁹-TNF, PSMA-ADC, DI-Leu16-IL2, SAR3419, SGN-35, or CMC544.In some embodiments, the targeting moiety may comprise, consist of, orconsist essentially of the antibody portion of HuM195-Ac-225,HuM195-Bi-213, Anyara (naptumomab estafenatox; ABR-217620), AS1409,Zevalin (ibritumomab tiuxetan), BIIB015, BT-062, Neuradiab, CDX-1307,CR011-vcMMAE, Trastuzumab-DM1 (R3502), Bexxar (tositumomab), IMGN242,IMGN388, IMGN901, ¹³¹I-labetuzumab, IMMU-102 (⁹⁰Y-epratuzumab), IMMU-107(⁹⁰Y-clivatuzumab tetraxetan), MDX-1203, CAT-8015, EMD 273063(hu14.18-IL2), Tucotuzumab celmoleukin (EMD 273066; huKS-IL2),¹⁸⁸Re-PTI-6D2, Cotara, L¹⁹-IL2, Teleukin (F16-IL2), Tenarad (F16-¹³¹I),L¹⁹-¹³¹I, L¹⁹-TNF, PSMA-ADC, DI-Leu16-IL2, SAR3419, SGN-35, or CMC544.

In some embodiments, the targeting moiety may be Brentuximab vedotin,Trastuzumab emtansine, Inotuzumab ozogamicin, Lorvotuzumab mertansine,Glembatumumab vedotin, SAR3419, Moxetumomab pasudotox, Moxetumomabpasudotox, AGS-16M8F, AGS-16M8F, BIIB-015, BT-062, IMGN-388, orIMGN-388.

In some embodiments, the targeting moiety may comprise, consist of, orconsist essentially of the antibody portion of Brentuximab vedotin,Trastuzumab emtansine, Inotuzumab ozogamicin, Lorvotuzumab mertansine,Glembatumumab vedotin, SAR3419, Moxetumomab pasudotox, Moxetumomabpasudotox, AGS-16M8F, AGS-16M8F, BIIB-015, BT-062, IMGN-388, orIMGN-388.

In some embodiments, the targeting moiety may comprise, consist of, orconsist essentially of Brentuximab, Inotuzumab, Gemtuzumab, Milatuzumab,Trastuzumab, Glembatumomab, Lorvotuzumab, or Labestuzumab.

As used herein, the term “linker” refers to a moiety that connects twoor more components to each other.

In some embodiments, the linker may be a linker disclosed in Janthur etal., “Drug Conjugates Such as Antibody Drug Conjugates (ADCs),Immunotoxins and Immunoliposomes Challenge Daily Clinical Practice,”Int. J. Mol. Sci. 2012, 13, 16020-16045. In some embodiments, the linkermay be a linker disclosed in Trail, P A, “Antibody Drug Conjugates asCancer Therapeutics,” Antibodies 2013, 2, 113-129. In some embodiments,the linker may be a linker disclosed in U.S. Pat. No. 7,829,531.

In some embodiments, the linker may comprise, consist of, or consistessentially of the linker portion of HuM195-Ac-225, HuM195-Bi-213,Anyara (naptumomab estafenatox; ABR-217620), AS1409, Zevalin(ibritumomab tiuxetan), BIIB015, BT-062, Neuradiab, CDX-1307,CR011-vcMMAE, Trastuzumab-DM1 (R3502), Bexxar (tositumomab), IMGN242,IMGN388, IMGN901, ¹³¹I-labetuzumab, IMMU-102 (⁹⁰Y-epratuzumab), IMMU-107(⁹⁰Y-clivatuzumab tetraxetan), MDX-1203, CAT-8015, EMD 273063(hu14.18-IL2), Tucotuzumab celmoleukin (EMD 273066; huKS-IL2),¹⁸⁸Re-PTI-6D2, Cotara, L¹⁹-IL2, Teleukin (F16-IL2), Tenarad (F16-¹³¹I),L¹⁹-¹³¹I, L¹⁹-TNF, PSMA-ADC, DI-Leu16-IL2, SAR3419, SGN-35, or CMC544.

In some embodiments, the linker may comprise, consist of, or consistessentially of the linker portion of Brentuximab vedotin, Trastuzumabemtansine, Inotuzumab ozogamicin, Lorvotuzumab mertansine, Glembatumumabvedotin, SAR3419, Moxetumomab pasudotox, Moxetumomab pasudotox,AGS-16M8F, AGS-16M8F, BIIB-015, BT-062, IMGN-388, or IMGN-388.

As will be appreciated by those of skill in the art, the above-definedcategories are not mutually exclusive. Thus, amino acids having sidechains exhibiting two or more physical-chemical properties can beincluded in multiple categories. For example, amino acid side chainshaving aromatic moieties that are further substituted with polarsubstituents, such as Tyr (Y), may exhibit both aromatic hydrophobicproperties and polar or hydrophilic properties, and can therefore beincluded in both the aromatic and polar categories. The appropriatecategorization of any amino acid will be apparent to those of skill inthe art, especially in light of the detailed disclosure provided herein.

While the above-defined categories have been exemplified in terms of thegenetically encoded amino acids, the amino acid substitutions need notbe, and in certain embodiments preferably are not, restricted to thegenetically encoded amino acids. In some embodiments, the activeagent-conjugate may contain genetically non-encoded amino acids. Thus,in addition to the naturally occurring genetically encoded amino acids,amino acid residues in the active agent-conjugate may be substitutedwith naturally occurring non-encoded amino acids and synthetic aminoacids.

Certain commonly encountered amino acids which provide usefulsubstitutions for the active agent-conjugates include, but are notlimited to, β-alanine (β-Ala) and other omega-amino acids such as3-aminopropionic acid, 2,3-diaminopropionic acid (Dpr), 4-aminobutyricacid and so forth; α-aminoisobutyric acid (Aib); ε-aminohexanoic acid(Aha); S-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly);ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA);t-butylglycine (t-BuG); N-methylisoleucine (MeIle); phenylglycine (Phg);cyclohexylalanine (Cha); norleucine (Nle); naphthylalanine (Nal);4-phenylphenylalanine, 4-chlorophenylalanine (Phe(4-Cl));2-fluorophenylalanine (Phe(2-F)); 3-fluorophenylalanine (Phe(3-F));4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen);1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic);β-2-thienylalanine (Thi); methionine sulfoxide (MSO); homoarginine(hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid (Dbu);2,3-diaminobutyric acid (Dab); p-aminophenylalanine (Phe (pNH₂));N-methyl valine (MeVal); homocysteine (hCys), homophenylalanine (hPhe)and homoserine (hSer); hydroxyproline (Hyp), homoproline (hPro),N-methylated amino acids and peptoids (N-substituted glycines).

Other amino acid residues not specifically mentioned herein can bereadily categorized based on their observed physical and chemicalproperties in light of the definitions provided herein.

The classifications of the genetically encoded and common non-encodedamino acids according to the categories defined above are summarized inTable 2, below. It is to be understood that Table 2 is for illustrativepurposes only and does not purport to be an exhaustive list of aminoacid residues and derivatives that can be used to substitute the activeagent-conjugate described herein.

TABLE 2 CLASSIFICATIONS OF COMMONLY ENCOUNTERED AMINO ACIDS GeneticallyClassification Encoded Non-Genetically Encoded Hydrophobic Aromatic F,Y, W Phg, Nal, Thi, Tic, Phe (4-Cl), Phe (2-F), Phe (3-F), Phe (4-F),hPhe Nonpolar L, V, I, M, G, t-BuA, t-BuG, MeIle, Nle, MeVal, Cha, A, PMcGly, Aib Aliphatic A, V, L, I b-Ala, Dpr, Aib, Ahx, MeGly, t-BuA,t-BuG, MeIle, Cha, Nle, MeVal Hydrophilic Acidic D, E Basic H, K, R Dpr,Orn, hArg, Phe(p-NH2), Dbu, Dab Polar C, Q, N, S. T Cit, AcLys, MSO,bAla, hSer Helix-Breaking P, G D-Pro and other D-amino acids (inL-peptides)

Other amino acid residues not specifically mentioned herein can bereadily categorized based on their observed physical and chemicalproperties in light of the definitions provided herein.

While in most instances, the amino acids of the compound-conjugate willbe substituted with L¹-enantiomeric amino acids, the substitutions arenot limited to L¹-enantiomeric amino acids. In some embodiments, thepeptides may advantageously be composed of at least one D-enantiomericamino acid. Peptides containing such D-amino acids are thought to bemore stable to degradation in the oral cavity, gut or serum than arepeptides composed exclusively of L¹-amino acids.

Examples of compounds of Formula I include, but are not limited to, thefollowing compounds:

-   -   or pharmaceutically acceptable salts or solvates thereof.

Pharmaceutical Compositions

In some embodiments, the compounds disclosed herein are used inpharmaceutical compositions. The compounds can be used, for example, inpharmaceutical compositions comprising a pharmaceutically acceptablecarrier prepared for storage and subsequent administration. Also,embodiments relate to a pharmaceutically effective amount of theproducts and compounds disclosed above in a pharmaceutically acceptablecarrier or diluent. Acceptable carriers or diluents for therapeutic useare well known in the pharmaceutical art, and are described, forexample, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985), which is incorporated herein by reference in itsentirety. Preservatives, stabilizers, dyes and even flavoring agents canbe provided in the pharmaceutical composition. For example, sodiumbenzoate, ascorbic acid and esters of p-hydroxybenzoic acid can be addedas preservatives. In addition, antioxidants and suspending agents can beused.

The compositions can be formulated and used as tablets, capsules, orelixirs for oral administration; suppositories for rectaladministration; sterile solutions, suspensions for injectableadministration; patches for transdermal administration, and sub-dermaldeposits and the like. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspension in liquid prior to injection, or asemulsions. Suitable excipients are, for example, water, saline,dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate,cysteine hydrochloride, and the like. In addition, if desired, theinjectable pharmaceutical compositions may contain minor amounts ofnontoxic auxiliary substances, such as wetting agents, pH bufferingagents, and the like. If desired, absorption enhancing preparations (forexample, liposomes), can be utilized.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds can be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or other organic oilssuch as soybean, grapefruit or almond oils, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents can beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions can be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments can be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses. For this purpose, concentratedsugar solutions can be used, which may optionally contain gum arabic,talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments can be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses.

To formulate the compounds of Formulae I and II as an anti-cancer agent,known surface active agents, excipients, smoothing agents, suspensionagents and pharmaceutically acceptable film-forming substances andcoating assistants, and the like can be used. Preferably alcohols,esters, sulfated aliphatic alcohols, and the like can be used as surfaceactive agents; sucrose, glucose, lactose, starch, crystallizedcellulose, mannitol, light anhydrous silicate, magnesium aluminate,magnesium methasilicate aluminate, synthetic aluminum silicate, calciumcarbonate, sodium acid carbonate, calcium hydrogen phosphate, calciumcarboxymethyl cellulose, and the like can be used as excipients;magnesium stearate, talc, hardened oil and the like can be used assmoothing agents; coconut oil, olive oil, sesame oil, peanut oil, soyacan be used as suspension agents or lubricants; cellulose acetatephthalate as a derivative of a carbohydrate such as cellulose or sugar,or methylacetate-methacrylate copolymer as a derivative of polyvinyl canbe used as suspension agents; and plasticizers such as ester phthalatesand the like can be used as suspension agents. In addition to theforegoing preferred ingredients, sweeteners, fragrances, colorants,preservatives and the like can be added to the administered formulationof the compound produced by the method of the embodiment, particularlywhen the compound is to be administered orally.

When used as an anti-cancer compound, for example, the compounds ofFormulae I and II or compositions including compounds of Formulae I andII can be administered by either oral or non-oral pathways. Whenadministered orally, it can be administered in capsule, tablet, granule,spray, syrup, or other such form. When administered non-orally, it canbe administered as an aqueous suspension, an oily preparation or thelike or as a drip, suppository, salve, ointment or the like, whenadministered via injection, subcutaneously, intraperitoneally,intravenously, intramuscularly, or the like.

In one embodiment, the anti-cancer agent can be mixed with additionalsubstances to enhance their effectiveness.

Methods of Administration

In an alternative embodiment, the disclosed compounds and the disclosedpharmaceutical compositions are administered by a particular method asan anti-cancer, or anti-inflammatory agent. Such methods include, amongothers, (a) administration though oral pathways, which administrationincludes administration in capsule, tablet, granule, spray, syrup, orother such forms; (b) administration through non-oral pathways, whichadministration includes administration as an aqueous suspension, an oilypreparation or the like or as a drip, suppository, salve, ointment orthe like; administration via injection, subcutaneously,intraperitoneally, intravenously, intramuscularly, intradermally, or thelike; as well as (c) administration topically, (d) administrationrectally, or (e) administration vaginally, as deemed appropriate bythose of skill in the art for bringing the compound of the presentembodiment into contact with living tissue; and (f) administration viacontrolled released formulations, depot formulations, and infusion pumpdelivery. As further examples of such modes of administration and asfurther disclosure of modes of administration, disclosed herein arevarious methods for administration of the disclosed compounds andpharmaceutical compositions including modes of administration throughintraocular, intranasal, and intraauricular pathways.

The pharmaceutically effective amount of the compositions that includethe described compounds required as a dose will depend on the route ofadministration, the type of animal, including human, being treated, andthe physical characteristics of the specific animal under consideration.The dose can be tailored to achieve a desired effect, but will depend onsuch factors as weight, diet, concurrent medication and other factorswhich those skilled in the medical arts will recognize. In a typicalembodiment, a compound represented by Formulae I and II can beadministered to a patient in need of an anti-cancer agent, until theneed is effectively reduced or preferably removed.

In practicing the methods of the embodiment, the products orcompositions can be used alone or in combination with one another, or incombination with other therapeutic or diagnostic agents. These productscan be utilized in vivo, ordinarily in a mammal, preferably in a human,or in vitro. In employing them in vivo, the products or compositions canbe administered to the mammal in a variety of ways, includingparenterally, intravenously, subcutaneously, intramuscularly,colonically, rectally, vaginally, nasally or intraperitoneally,employing a variety of dosage forms. Such methods may also be applied totesting chemical activity in vivo.

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and mammalian species treated,the particular compounds employed, and the specific use for which thesecompounds are employed. The determination of effective dosage levels,that is the dosage levels necessary to achieve the desired result, canbe accomplished by one skilled in the art using routine pharmacologicalmethods. Typically, human clinical applications of products arecommenced at lower dosage levels, with dosage level being increaseduntil the desired effect is achieved. Alternatively, acceptable in vitrostudies can be used to establish useful doses and routes ofadministration of the compositions identified by the present methodsusing established pharmacological methods.

In non-human animal studies, applications of potential products arecommenced at higher dosage levels, with dosage being decreased until thedesired effect is no longer achieved or adverse side effects disappear.The dosage may range broadly, depending upon the desired affects and thetherapeutic indication. Typically, dosages can be between about 10 mg/kgand 100 mg/kg body weight, preferably between about 100 mg/kg and 10mg/kg body weight. Alternatively dosages can be based and calculatedupon the surface area of the patient, as understood by those of skill inthe art. Administration is preferably oral on a daily or twice dailybasis.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. See forexample, Fingl et al., in The Pharmacological Basis of Therapeutics,1975, which is incorporated herein by reference in its entirety. Itshould be noted that the attending physician would know how to and whento terminate, interrupt, or adjust administration due to toxicity, or toorgan dysfunctions. Conversely, the attending physician would also knowto adjust treatment to higher levels if the clinical response were notadequate (precluding toxicity). The magnitude of an administrated dosein the management of the disorder of interest will vary with theseverity of the condition to be treated and to the route ofadministration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above can be used in veterinary medicine.

Depending on the specific conditions being treated, such agents can beformulated and administered systemically or locally. A variety oftechniques for formulation and administration can be found inRemington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co.,Easton, Pa. (1990), which is incorporated herein by reference in itsentirety. Suitable administration routes may include oral, rectal,transdermal, vaginal, transmucosal, or intestinal administration;parenteral delivery, including intramuscular, subcutaneous,intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections.

For injection, the agents of the embodiment can be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer. Forsuch transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art. Use of pharmaceutically acceptable carriersto formulate the compounds herein disclosed for the practice of theembodiment into dosages suitable for systemic administration is withinthe scope of the embodiment. With proper choice of carrier and suitablemanufacturing practice, the compositions disclosed herein, inparticular, those formulated as solutions, can be administeredparenterally, such as by intravenous injection. The compounds can beformulated readily using pharmaceutically acceptable carriers well knownin the art into dosages suitable for oral administration. Such carriersenable the compounds of the embodiment to be formulated as tablets,pills, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for oral ingestion by a patient to be treated.

Agents intended to be administered intracellularly can be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents can be encapsulated into liposomes, thenadministered as described above. All molecules present in an aqueoussolution at the time of liposome formation are incorporated into theaqueous interior. The liposomal contents are both protected from theexternal micro-environment and, because liposomes fuse with cellmembranes, are efficiently delivered into the cell cytoplasm.Additionally, due to their hydrophobicity, small organic molecules canbe directly administered intracellularly.

Determination of the effective amounts is well within the capability ofthose skilled in the art, especially in light of the detailed disclosureprovided herein. In addition to the active ingredients, thesepharmaceutical compositions may contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically. The preparations formulated for oraladministration can be in the form of tablets, dragees, capsules, orsolutions. The pharmaceutical compositions can be manufactured in amanner that is itself known, for example, by means of conventionalmixing, dissolving, granulating, dragee-making, levitating, emulsifying,encapsulating, entrapping, or lyophilizing processes.

Compounds disclosed herein can be evaluated for efficacy and toxicityusing known methods. For example, the toxicology of a particularcompound, or of a subset of the compounds, sharing certain chemicalmoieties, can be established by determining in vitro toxicity towards acell line, such as a mammalian, and preferably human, cell line. Theresults of such studies are often predictive of toxicity in animals,such as mammals, or more specifically, humans. Alternatively, thetoxicity of particular compounds in an animal model, such as mice, rats,rabbits, dogs or monkeys, can be determined using known methods. Theefficacy of a particular compound can be established using several artrecognized methods, such as in vitro methods, animal models, or humanclinical trials. Art-recognized in vitro models exist for nearly everyclass of condition, including the conditions abated by the compoundsdisclosed herein, including cancer, cardiovascular disease, and variousimmune dysfunction, and infectious diseases. Similarly, acceptableanimal models can be used to establish efficacy of chemicals to treatsuch conditions. When selecting a model to determine efficacy, theskilled artisan can be guided by the state of the art to choose anappropriate model, dose, and route of administration, and regime. Ofcourse, human clinical trials can also be used to determine the efficacyof a compound in humans.

As will be understood by one of skill in the art, “need” is not anabsolute term and merely implies that the patient can benefit from thetreatment of the anti-cancer agent in use. By “patient” what is meant isan organism that can benefit by the use of an anti-cancer agent.

“Therapeutically effective amount,” “pharmaceutically effective amount,”or similar term, means that amount of drug or pharmaceutical agent thatwill result in a biological or medical response of a cell, tissue,system, animal, or human that is being sought. In a preferredembodiment, the medical response is one sought by a researcher,veterinarian, medical doctor, or other clinician.

In one embodiment, a described compound, preferably a compound of anyone of Formula I and/or II, including those as described herein, isconsidered an effective anti-cancer agent if the compound can influence10% of the cancer cells, for example. In a more preferred embodiment,the compound is effective if it can influence 10 to 50% of the cancercells. In an even more preferred embodiment, the compound is effectiveif it can influence 50-80% of the cancer cells. In an even morepreferred embodiment, the compound is effective if it can influence80-95% of the cancer cells. In an even more preferred embodiment, thecompound is effective if it can influence 95-99% of the cancer cells.“Influence” is defined by the mechanism of action for each compound.

Some embodiments include methods of delivering a drug molecule to an invivo mammalian cell by administering to the mammal an antibody-drugconjugate comprising a drug moiety. In some embodiments theadministration is parenteral (e.g., intravenously). In otherembodiments, the administration is oral. In some embodiments, thatantibody-drug conjugate comprises an antibody, a chemical linker asdescribed herein, and the drug moiety.

Methods of Treatment

Some embodiments include methods of treating cancer by administering acompound of any one of Formula I and/or II to a subject in need ofcancer therapy. Non-limiting cancers that can be treated using thecompounds described herein include bladder cancer, breast cancer, coloncancer, rectal cancer, endometrial cancer, kidney cancer, lung cancer,melanoma, non-Hodgkin lymphoma, glioblastoma, pancreatic cancer,prostate cancer, and thyroid cancer.

Some embodiments include the treatment of cancer including, but notlimited to a carcinoma, a sarcoma, a lymphoma, a leukemia, and ablastoma. Non-limiting cancers that can be treated using the compoundsdescribed herein include bladder cancer, breast cancer, colon cancer,rectal cancer, endometrial cancer, kidney cancer, lung cancer, melanoma,non-Hodgkin lymphoma, leukemia, glioblastoma, pancreatic cancer,prostate cancer, and thyroid cancer.

Some embodiments include the treatment of cancer including, but notlimited to a carcinoma, a sarcoma, a lymphoma, a leukemia, and ablastoma. Non-limiting cancers that can be treated using the compoundsdescribed herein include bladder cancer, breast cancer, colon cancer,rectal cancer, endometrial cancer, kidney cancer, lung cancer, melanoma,non-Hodgkin lymphoma, leukemia, glioblastoma, pancreatic cancer,prostate cancer, and thyroid cancer.

Some embodiments provide a method of treating a cancer comprisingadministering a compound of Formula I or II, or a pharmaceuticallyacceptable salt thereof to a subject in need thereof. In someembodiments, the cancer is leukemia. In some embodiments, the cancer ismultiple myeloma.

Some embodiments provide a method of treating melanoma, comprisingadministering a compound of Formula I or II, or a pharmaceuticallyacceptable salt thereof to a subject in need thereof. Some embodimentsprovide a method of treating multiple myeloma, comprising administeringa compound of Formula I or II, or a pharmaceutically acceptable saltthereof to a subject in need thereof.

Some embodiments provide a use of a compound of Formula II to provide achemical entity to a target location, where the chemical entity has thefollowing structure:

-   -   or pharmaceutically acceptable salts or solvates thereof.

In some embodiments, the subject is a human.

Some embodiments provide a method delivering a compound having thestructure of Formula I to an in vivo mammalian cell, the methodcomprising administering a compound having the structure of Formula IIto a mammal comprising the in vivo mammalian cell. In some embodiments,the antibody-drug conjugate is administered parenterally. In someembodiments, the antibody-drug conjugate is administered intravenously.In some embodiments, the antibody-drug conjugate is administered orally.

Some embodiments provide use of a compound having the structure ofFormula II to provide a compound having the structure of Formula I to atarget location.

Further embodiments include administering a combination of compounds toa subject in need thereof. A combination can include a compound,composition, pharmaceutical composition described herein with anadditional medicament.

Some embodiments include co-administering a compound, composition,and/or pharmaceutical composition described herein, with an additionalmedicament. By “co-administration,” it is meant that the two or moreagents may be found in the patient's bloodstream at the same time,regardless of when or how they are actually administered. In oneembodiment, the agents are administered simultaneously. In one suchembodiment, administration in combination is accomplished by combiningthe agents in a single dosage form. In another embodiment, the agentsare administered sequentially. In one embodiment the agents areadministered through the same route, such as orally. In anotherembodiment, the agents are administered through different routes, suchas one being administered orally and another being administered i.v.

The term “patient” includes human and animal subjects.

The term “contacting” refers to bringing two or more materials intoclose enough proximity that they may interact. In certain embodiments,contacting can be accomplished in a vessel such as a test tube, a petridish, or the like. In certain embodiments, contacting may be performedin the presence of additional materials. In certain embodiments,contacting may be performed in the presence of cells. In certain of suchembodiments, one or more of the materials that are being contacted maybe inside a cell. Cells may be alive or may dead. Cells may or may notbe intact.

EXAMPLES

The following examples are set forth merely to assist in understandingthe embodiments and should not be construed as limiting the embodimentsdescribed and claimed herein in any way. Variations of the invention,including the substitution of all equivalents now known or laterdeveloped, which would be within the purview of those skilled in theart, and changes in formulation or minor changes in experimental design,are to be considered to fall within the scope of the inventionincorporated herein.

Example 1 General Procedure A—Synthesis of Compounds of Formula I

A compound of General Formula I may be synthesized following proceduresknown in the art with appropriate modifications such as disclosed inHamada et al., “Efficient stereoselective synthesis of dolastatin 10, anantineoplastic peptide from a sea hare,” Tetrahedron Lett., 1991, 32(7):931-934. For example, a compound of General Formula I-A may be treatedwith TFA to afford a secondary amine intermediate that may be reactedwith a compound of General Formula A under coupling conditions, such asBopCl, NEt₃/CH₂C₂, to afford a compound of General Formula II-A. Acompound of General Formula II-A may be then treated with TFA to afforda primary amine intermediate that may be reacted with a compound ofGeneral Formula B under coupling conditions, such as DEPC, NEt₃/DMF, toafford a compound of General Formula I.

Synthesis of General Formula A:

Synthesis of Amino Acid with Olefin Intermediate:

Compound 11 may be made according to the procedure disclosed in Voica,et al., “Guided desaturation of unactivated aliphatics,” NatureChemistry, 2012, 4: 629-635.

Compound 13 may be synthesized following procedures known in the artwith appropriate modifications such as disclosed in Petasis, et al., “ANew and Practical Synthesis of α-Amino Acids from Alkenyl BoronicAcids,” J. Am. Chem. Soc., 1997, 119, 445-446. Compound 13 may befurther functionalized by methods known in the art to afford a compoundof General Formula IB-1. The compound of General Formula IB-1 may besubjected to hydrogenation to afford a compound of General Formula IB-2.The compound of General Formula IB-2 may be treated with (Boc)₂O underthe appropriate conditions to afford a compound of General Formula A.

Synthesis of halogen Compounds of General Formula A:

A compound of General Formula IC-2 (where PG is a protecting group suchas Boc or as shown in Scheme IA and Scheme IB) may be synthesized from acompound of General Formula IC-1 (where PG is a protecting group such asBoc or as shown in Scheme IA and Scheme IB), for example compound 11 orcompound 13, following procedures known in the art with appropriatemodifications such as disclosed in Barker, et al.,“Fe(III)/NaBH₄-Mediated Free Radical Hydrofluorination of UnactivatedAlkenes,” J. Am. Chem. Soc., 2012, 134, 13588-13591 (X¹═F) or Gaspar, etal., “Catalytic Hydrochlorination of Unactivated Olefins withpara-Toluenesulfonyl Chloride,” Angew. Chem. Int. Ed, 2008, 47,5778-5760 (X¹═Cl).

Synthesis of azide or amine Compounds of General Formula A:

A compound of General Formula ID-1 (where PG is a protecting group suchas Boc) or General Formula ID-2 (where PG is a protecting group such asBoc) may be synthesized from a compound of General Formula IC-1, forexample compound 11 or compound 13, following procedures known in theart with appropriate modifications such as disclosed in Leggans, et al.,“Iron(III)/NaBH4-Mediated Additions to Unactivated Alkenes: Synthesis ofNovel 20′-Vinblastine Analogues,” Org. Lett., 2012, 14, 1428-1431 orWaser, et al., “Cobalt-Catalyzed Hydroazidation of Olefins: ConvenientAccess to Alkyl Azides,” J. Am. Chem. Soc., 2005, 127, 8294-8295.

Synthesis of hydroxy Compounds of General Formula A:

A compound of General Formula IE-2 may be synthesized from a compound ofGeneral Formula IE-1 following the procedure disclosed in Yonezawa, etal., “Facile Synthesis of L-3,4-Didehydrovaline Constituting anAntibiotic, Phomopsin A,” Synthesis, 2000, 5, 634-636.

Compounds 15 and 16 may be synthesized from compound 14 followingprocedures known in the art.

Synthesis of cyano Compounds of General Formula A:

A compound of General Formula IG-1 (where PG is a protecting group suchas Boc) may be synthesized from a compound of General Formula IC-1following procedures known in the art with appropriate modificationssuch as disclosed in Gaspar, et al., “Catalytic Hydrochlorination ofUnactivated Olefins with para-Toluenesulfonyl Chloride,” Angew. Chem.Int. Ed, 2008, 47, 5778-5760 and references cited therein.

Synthesis of Compounds of General Formula G:

Compounds of General Formula G may be synthesized from a compound ofGeneral Formula IH-1 following procedures known in the art withappropriate modifications.

Synthesis of Compound 101

Compound 101 was synthesized using a procedure similar to Scheme I-Aabove and following procedures known in the art with appropriatemodifications such as disclosed in Hamada et al., “Efficientstereoselective synthesis of dolastatin 10, an antineoplastic peptidefrom a sea hare,” Tetrahedron Lett., 1991, 32(7): 931-934. For example,compound IA may be treated with TFA to afford a secondary amineintermediate that may be reacted with a compound C under couplingconditions, such as BopCl, NEt₃/CH₂Cl₂, to afford compound IIA. CompoundIIA may be then treated with TFA to afford a primary amine intermediatethat may be reacted with compound D under coupling conditions, such asDEPC, NEt₃/DMF, to afford a compound 101. The MS2 fragmentation patternof compound 101 was measured and determined to be similar to that ofDolastatin 10 and Symplostatin-1, which confirms the structure ofcompound 101.

Synthesis of Compound 102

Compound 102 may be synthesized following procedures known in the artwith appropriate modifications such as disclosed in Hamada et al.,“Efficient stereoselective synthesis of dolastatin 10, an antineoplasticpeptide from a sea hare,” Tetrahedron Lett., 1991, 32(7): 931-934. Forexample, compound IIA may be then treated with TFA to afford a primaryamine intermediate that may be reacted with compound E under couplingconditions, such as DEPC, NEt₃/DMF, to afford a compound 102.

Synthesis of Compound 103

Compound 103 may be synthesized following procedures known in the artwith appropriate modifications such as disclosed in Hamada et al.,“Efficient stereoselective synthesis of dolastatin 10, an antineoplasticpeptide from a sea hare,” Tetrahedron Lett., 1991, 32(7): 931-934. Forexample, compound IA may be treated with TFA to afford a secondary amineintermediate that may be reacted with a compound F under couplingconditions, such as BopCl, NEt₃/CH₂Cl₂, to afford compound IIB. CompoundIIB may be then treated with TFA to afford an intermediate that may bereacted with compound D under coupling conditions, such as DEPC,NEt₃/DMF, to afford a compound 103.

Synthesis of Compound 101, 104, 105 and 106:

A reaction chamber equipped with magnetic stirbar was charged with IA1(0.1 mmol), Fmoc-(S)-amino acid (0.2 mmol, 2.0 equiv.), and CDMT(2-chloro-4,6-dimethoxy-1,3,5-triazine, 0.2 mmol, 2.0 equiv.). Thereaction chamber was flushed with Ar and the materials were dissolved inanhydrous THF (0.05 M) followed by slow addition of N-methylmorpholine(0.35 mmol, 3.5 equiv.) over 2 min. The pale yellow solution was allowedto stir at rt under Ar for 18 h. Subsequently, the crude mixture wasloaded onto HP20ss (2 g), and purified via CombiFlash Rf (C18 15.5 g, 30mL/min, CH₃CN/H₂O linear gradient from 35-100% CH₃CN over 12 min ramp).The desired products (IIA1a to IA1d) were isolated as a colorless oil(55-70% yields).

A reaction chamber equipped with magnetic stirbar was charged withFmoc-tetrapeptide, IIA1 (0.05 mmol). The material was dissolved in CH₃CN(0.01 M) followed by diethylamine (1.25 mmol, 25 equiv.) addition at rtand the resulting mixture was allowed to stir for 3 h. The mixture wasextracted with pentane (3×3 mL) and concentrated to yield crude materialas oil. The crude material was subjected to next step without furtherpurification (90-94% yields).

A reaction chamber equipped with magnetic stirbar was charged withtetrapeptide, IA2 (0.05 mmol) and N,N-dimethyl-L¹-valine (0.1 mmol, 2equiv.). The materials were dissolved in anhydrous DMF (0.05 M) andcooled in an ice bath (−5° C.) under Ar followed by triethylamine (0.11mmol, 2.2 equiv.) addition and the resulting mixture was allowed to stirfor 5 min at 5° C. A solution of HATU (0.075 mmol, 1.5 equiv.) inanhydrous DMF (0.25 M) was slowly added into the mixture at 5° C. Theresulting mixture was allowed to stir in an ice bath for additional 45min. The mixture was then loaded onto HP20ss (2 g) and purified viaCombiFlash Rf (C18 5.5 g, 18 mL/min, CH₃CN/H₂O linear gradient from30-100% CH₃CN over 14 min ramp). The desired products were isolated asan amorphous solid (80-90% yields). Observed ESI HRMS for compound 101:m/z 801.49798 [M+H]+, compound 104: m/z 787.4815 [M+H]+, compound 105:m/z 773.4662 [M+H]+, and compound 106: m/z 787.4790 [M+H]+.

Synthesis of Compound 107:

A reaction chamber equipped with magnetic stirbar was charged with IA1(90 mg, 0.161 mmol), Fmoc-(S)-2-amino-3-hydroxy-3-methylbutanoic acid(114 mg, 0.322 mmol, 2.0 equiv.), and CDMT(2-chloro-4,6-dimethoxy-1,3,5-triazine, 57 mg, 0.322 mmol, 2.0 equiv.).The reaction chamber was flushed with Ar and the materials weredissolved in anhydrous THF (5.0 mL, 0.03 M) followed by slow addition ofN-methylmorpholine (62 μL, 0.564 mmol, 3.5 equiv.) over 2 min. The paleyellow solution was allowed to stir at r under Ar for 18 h.Subsequently, the crude mixture was loaded onto HP20ss (2.5 g), andpurified via CombiFlash Rf (C18 15.5 g, 30 mL/min, CH₃CN/H₂O lineargradient from 35-100% CH₃CN over 12 min ramp). The desired product wasisolated as a colorless oil (77 mg, 0.086 mmol, 53%).

A reaction chamber equipped with magnetic stirbar was charged withFmoc-tetrapeptide IIA1a (75 mg, 0.084 mmol). The material was dissolvedin CH₃CN (4.5 mL) followed by diethylamine (0.219 mL, 2.092 mmol, 25equiv.) addition at rt and the resulting mixture was allowed to stir for3 h. The mixture was extracted with pentane (3×3 mL) and concentrated toyield crude material as oil. The crude material was subjected to nextstep without further purification (53 mg, 0.078 mmol, 94%).

A reaction chamber equipped with magnetic stirbar was charged with freebase tetrapeptide (IIA2a, 50 mg, 0.074 mmol) andFmoc-2-methylaminoisobutyric acid (50 mg, 0.148 mmol, 2 equiv.). Thematerials were dissolved in anhydrous DMF (1.25 mL) and cooled in an icebath (−5° C.) under Ar followed by triethylamine (23 μL, 0.163 mmol, 2.2equiv.) addition and the resulting mixture was allowed to stir for 5 minat 5° C. A solution of HATU (42 mg, 0.111 mmol, 1.5 equiv.) in anhydrousDMF (0.5 mL) was slowly added into the mixture at 5° C. The mixtureturned pale yellow and was allowed to stir in an ice bath for additional45 min. Subsequently, the crude mixture was loaded onto HP20ss (2.5 g)and purified via CombiFlash Rf (C18 5.5 g, 18 mL/min, CH₃CN/H₂O lineargradient from 30-100% CH₃CN over 14 min ramp). The desired product IIA3awas isolated as an amorphous solid (60 mg, 0.06 mmol, 82%).

A reaction chamber equipped with magnetic stirbar was charged withFmoc-pentapeptide, IIA3a (60 mg, 0.06 mmol). The material was dissolvedin CH₃CN (4.5 mL) and purged with Ar. Diethylamine (0.184 mL, 1.76 mmol,25 equiv.) was then added at rt and allowed to stir for 3 h. The crudemixture was then loaded onto HP20ss (2 g) and purified using CombiFlashRf (C18Aq 5.5 g, 18 mL/min, CH₃CN/H₂O linear gradient from 0-50% CH₃CNover 12 min ramp). The desired product, Compound 107 was isolated as acolorless glassy solid (40 mg, 0.052 mmol, 86%). Observed ESI HRMS: m/z773.4659 [M+H]+. Compound 107 exists as conformers in a 3:2 ratio inCD₃OD solvent; the NMR chemical shifts for the major conformer arelisted here. ¹H NMR (CD₃OD, 600 MHz): δ 7.79 (d, J=3.2 Hz, 1H), 7.53 (d,J=3.2 Hz, 1H), 7.31 (d, J=7.3 Hz, 2H), 7.25 (t, J=7.3 Hz, 2H), 7.21 (d,J=7.3 Hz, 1H), 5.66 (dd, J=4.1, 11.4 Hz, 1H), 5.02 (s, 1H), 4.15 (m,1H), 3.67 (m, 1H), 3.57 (m, 1H), 3.51 (dd, J=4.5, 14 Hz, 1H), 3.42 (d,J=10.8 Hz, 1H), 3.38 (s, 3H), 3.35 (s, 3H), 3.33 (m, 1H), 3.20 (m, 1H),3.20 (s, 3H), 3.04 (m, 1H), 2.62 (s, 3H), 2.48 (m, 1H), 2.38 (m, 1H),2.29 (m, 1H), 1.89 (m, 1H), 1.88 (m, 1H), 1.71 (m, 1H), 1.65 (s, 3H),1.60 (m, 1H), 1.57 (s, 3H), 1.40 (m, 1H), 1.39 (s, 3H), 1.37 (m, 1H),1.26 (s, 3H), 1.16 (d, J=6.6 Hz, 3H), 1.04 (m, 1H), 1.01 (d, J=7.3, 3H),0.87 (t, J=7.4, 3H). ¹³C NMR (CD₃0D, 150 MHz): S 175.0, 173.9, 173.4,172.0, 170.1, 143.5, 137.5, 129.8, 129.8, 128.7, 128.7, 127.7, 120.3,86.4, 73.0, 70.9, 63.1, 61.9, 60.3, 58.4, 58.3, 56.6, 53.0, 47.8, 45.3,41.3, 36.8, 33.6, 33.1, 28.1, 27.2, 26.7, 26.4, 25.7, 25.3, 21.8, 21.3,16.2, 14.8, 10.6.

Synthesis of Compound 108, 109 and 110:

A reaction chamber equipped with magnetic stirbar was charged with IB1(0.1 mmol), Fmoc-(S)-2-amino-3-hydroxy-3-methylbutanoic acid (0.2 mmol,2.0 equiv.), and CDMT (2-chloro-4,6-dimethoxy-1,3,5-triazine, 0.2 mmol,2.0 equiv.). The reaction chamber was flushed with Ar and the materialswere dissolved in anhydrous THF (0.05 M) followed by slow addition ofN-methylmorpholine (0.35 mmol, 3.5 equiv.) over 2 min. The pale yellowsolution was allowed to stir at rt under Ar for 18 h. Subsequently, thecrude mixture was loaded onto HP20ss (2 g), concentrated, and purifiedvia Combiflash Rf (C18 15.5 g, 30 ml/min, CH₃CN/H₂O linear gradient from35-100% CH₃CN over 12 min ramp). The desired product was isolated as acolorless oil (50% yield).

A reaction chamber equipped with magnetic stirbar was charged withFmoc-tetrapeptide (IIB, 0.05 mmol). The material was dissolved in CH₃CN(0.01 M) followed by diethylamine (1.25 mmol, 25 equiv.) addition at rtand the resulting mixture was allowed to stir for 3 h. The mixture wasextracted with pentane (3×3 mL) and concentrated to yield crude materialas oil. The crude material was subjected to next step without furtherpurification (70% yield).

A reaction chamber equipped with magnetic stirbar was charged with freebase tetrapeptide (IIB2, 0.05 mmol) and either A, B, or C amino acids(0.1 mmol, 2 equiv.). The materials were dissolved in an hydrous DMF(0.05M) and cooled in an ice bath (˜5° C.) under Ar followed bytriethylamine (0.11 mmol, 2.2 equiv.) addition and the resulting mixturewas allowed to stir for 5 min at 5° C. A solution of HATU (0.075 mmol,1.5 equiv.) in anhydrous DMF (0.25 M) was slowly added into mixture at5° C. The resulting mixture was allowed to stir in an ice bath foradditional 45 m. The mixture was then loaded onto HP2ss (2 g) andpurified via CombiFlash Rf (C18 5.5 g, 18 m min, CH₃CN/H₂O lineargradient from 30-100% CH₃CN over 14 min ramp). The desired products wereisolated as amorphous solids (80-90% yields). Observed ESI HRMS forcompound 108: m/z 748.5263 [M+H]⁺.

A reaction chamber equipped with magnetic stirbar was charged withFmoc-pentapeptide II or III (0.05 mmol). The material was dissolved inCH₃CN (0.01 M) followed by diethylamine (1.25 mmol, 25 equiv.) additionat rt and the resulting mixture was allowed to stir for 3h. The mixturewas extracted with pentane (3×3 mL) and concentrated to yield crudematerial as oil. The solution was then loaded onto HP20ss (2 g) andpurified using CombiFlash Rf (C18Aq 5.5 g, 18 mL/min, CH₃CN/H₂O lineargradient from 0-50% CH₃CN over 12 min ramp). The desired product wasisolated as a colorless oil (85-90% yields). Observed ESI HRMS forCompound 109: m/z 734.5100 [M+H]-; Compound 110: m/z 720.4948 [M+H]+

Synthesis of Compound 111:

A reaction chamber equipped with magnetic stirbar was charged with IA1(10 mg, 0.018 mmol), S-methyl-L¹-cysteine (13 mg, 0.036 mmol, 2.0equiv.), and CDMT (2-chloro-4,6-dimethoxy-1,3,5-triazine, 6.28 mg, 0.036mmol, 2.0 equiv.). The reaction chamber was flushed with Ar and thematerials were dissolved in anhydrous THF (360 μL, 0.05 M) followed byslow addition of N-methylmorpholine (6.89 μL, 0.063 mmol, 3.5 equiv.)over 2 min. The pale yellow solution was allowed to stir at r under Arfor 24 h. Subsequently, the crude mixture was concentrated, and purifiedvia CombiFlash Rf (C18Aq 30 g, 35 m/min, CH₃CN/H₂O linear gradient from0-100% CH₃CN over 14 min ramp). The desired product, IIA1e, was isolatedas a white powder (9.5 mg, 0.014 mmol, 79%). Observed ESI HRMS m/z898.4204 [M+H]⁺,

A reaction chamber equipped with magnetic stirbar was charged withFmoc-tetrapeptide, (IIA1e, 9.5 mg, 0.0106 mmol). The material wasdissolved in CH₃CN (1 mL) followed by diethylamine (0.4 mL, 3.83 mmol,362 equiv.) addition at rt and the resulting mixture was allowed to stirfor 0.5 h. The crude reaction was concentrated, and purified viaCombiFlash Rf (C18Aq 30 g, 35 mL/min, CH₃CN/H₂O linear gradient from0-100% CH₃CN over 12 min ramp). The desired product (IIA2e) was isolatedas a white solid (7 mg, 0.0104 mmol, 98%). Observed ESI HRMS m/z676.3533 [M+H]⁺.

A reaction chamber equipped with magnetic stirbar was charged with freebase tetrapeptide (IIA2e, 7 mg, 0.0104 mmol) and Fmoc-2-methylaminoisobutyric acid (7.03 mg, 0.021 mol, 2 equiv.). The materials weredissolved in anhydrous DMF (0.7 mL) and cooled in an ice bath (˜5° C.)under Ar followed by triethylamine (3.18 μL, 0.023 mmol, 2.2 equiv.)addition and the resulting mixture was allowed to stir for 5 min at 5°C. A solution of HATU (5.91 mg, 0.016 mmol, 1.5 equiv.) in anhydrous DMF(0.5 mL) was slowly added into the mixture at 5° C. The mixture turnedpale yellow and was allowed to stir in an ice bath for additional 45min. The mixture was concentrated and purified via CombiFlash Rf (C18Aq30 g, 35 mL/min, CH₃CN/H₂O linear gradient from 0-100% CH₃CN over 15minutes). The desired product (IIA3e) was isolated as an amorphous solid(11 mg, 0.011 mmol, 107%). Observed ESI HRMS m/z 997.4882 [M+H]+.

A reaction chamber equipped with magnetic stirbar was charged withFmoc-pentapeptide (IIA3e, 11 mg, 0.011 mmol). The material was dissolvedin CH₃CN (1.0 mL) and purged with Ar. Diethylamine (0.4 mL, 3.83 mmol,347 equiv.) was then added at rt and the resulting mixture allowed tostir for 0.5 h. The mixture was concentrated and purified usingCombiFlash Rf (C18Aq 30 g, 35 mL/min, CH₃CN/H₂O linear gradient from0-100% CH₃CN over 15 minutes). The desired product, Compound 111, wasisolated as a colorless oil (3.03 mg, 3.91 μmol, 35%). Observed ESI HRMSm/z 388.218 [M+2H]²⁺.

Synthesis of Compound 112:

A reaction chamber equipped with magnetic stirbar was charged with IA1(10 mg, 0.018 mmol), Fmoc-Dap(Boc)-OH (15 mg, 0.036 mmol, 2.0 equiv.),and CDMT (2-chloro-4,6-dimethoxy-1,3,5-triazine, 6.28 mg, 0.036 mmol,2.0 equiv.). The reaction chamber was flushed with Ar and the materialswere dissolved in anhydrous THF (180 μL, 0.05 M) followed by slowaddition of N-methylmorpholine (6.89 μL, 0.063 mmol, 3.5 equiv.) over 2min. The pale yellow solution was allowed to stir at r under Ar for 24h. Upon completion the crude reaction was concentrated, and purified viaCombiFlash Rf (C18Aq 30 g, 35 mL/min, CH₃CN/H₂O linear gradient from0-100% CH₃CN over 15 min ramp). The desired product was isolated as awhite powder (9 mg, 9.30 μmol, 52%). Observed ESI HRMS m/z 967.4962[M+H]+.

To a reaction chamber equipped with magnetic stirbar was charged withFmoc-tetrapeptide (IIA1f, 9 mg, 9.30 μmol). The material was dissolvedin CH₃CN (1 mL) followed by diethylamine (0.4 mL, 3.81 mmol, 410 equiv.)addition at rt and reaction mixture was allowed to stir for 0.5 h. Thecrude reaction was concentrated, and purified via CombiFlash Rf (C18Aq30 g, 30 mL/min, CH₃CN/H₂O linear gradient from 0-100% CH₃CN over 12 minramp). The desired product was isolated as a white powder (8 mg, 9.30μmol, 115%). Observed ESI HRMS m/z 745.4278 [M+H]⁺.

A reaction chamber equipped with magnetic stirbar was charged with freebase tetrapeptide (IIA2f, 8 mg, 0.0107 mmol) and Fmoc-2-methylaminoisobutyric acid (7.29 mg, 0.0201 mol, 2 equiv.). The materials weredissolved in anhydrous DMF (0.7 mL) and cooled in an ice bath (−5° C.)under Ar followed by triethylamine (3.29 μL, 0.024 mmol, 2.2 equiv.)addition and the reaction was allowed to stir for 5 min at 5° C. Asolution of HATU (6.12 mg, 0.016 mmol, 1.5 equiv.) in anhydrous DMF (0.5mL) was slowly added into reaction mixture at 5° C. The reaction turnedpale yellow and was allowed to stir in an ice bath for additional 45min. The reaction solution was concentrated and purified via CombiFlashRf (C18Aq 30 g, 35 mL/min, CH₃CN/H₂O linear gradient from 0-100% CH₃CNover 15 minutes). The desired product was isolated as a white powder(3.8 mg, 3.56 μmol, 33%). Observed ESI HRMS m/z 1066.5639 [M+H]⁺.

A reaction chamber equipped with magnetic stirbar was charged withFmoc-pentapeptide (IIA3f, 3.8 mg, 3.56 μmol). The material was dissolvedin CH₃CN (1.0 mL) and purged with Ar. Diethylamine (0.406 mL, 3.88 mmol,1090 eq) was then added at rt and allowed to stir for 0.5 h. Thesolution was concentrated and purified using CombiFlash Rf (C18Aq 30 g,35 mL/min, CH₃CN/H₂O linear gradient from 0-100% CH₃CN over 15 minutes).The desired product was isolated as white powder (2.1 mg, 2.488 μmol,70%). Observed ESI HRMS m/z 422.7568 [M+2H]²⁺.

A reaction chamber equipped with a magnetic stirbar was charged withBoc-pentapeptide (IIA4f, 2.1 mg, 2.488 μmol). The material was dissolvedin CH₂C₂ (1 mL) followed by the addition of trifluoroacetic acid (0.180mL, 2.339 mmol, 940 eq) at 0° C. and allowed to stir at 0° C. for 0.5 h,then warmed to rt and allowed to stir for an additional 0.5 h. Thesolution was concentrated and purified using CombiFlash Rf (C18Aq 30 g,35 mL/min, CH₃CN/H₂O linear gradient from 0-100% CH₃CN over 15 minutes).The desired product was isolated as a white powder (350 μg, 2.488 μmol,20%). Observed ESI HRMS m/z 744.4502 [M+H]⁺.

Synthesis of Compound 113:

A reaction chamber equipped with magnetic stirbar was charged withtetrapeptide (IIA2a, 3 mg, 4.45 μmol) and Fmoc-2-methylamino isobutyricacid (2.90 mg, 8.90 μmol, 2 equiv.). The materials were dissolved inanhydrous DMF (0.7 mL) and cooled in an ice bath (−5° C.) under Arfollowed by triethylamine (1.365 μL, 9.79 μmol, 2.2 equiv.) addition andthe resulting mixture was allowed to stir for 5 min at 5° C. A solutionof HATU (2.54 mg, 6.68 μmol, 1.5 equiv.) in anhydrous DMF (0.5 mL) wasslowly added into the mixture at 5° C. The mixture turned pale yellowand was allowed to stir in an ice bath for additional 45 min. Themixture was concentrated and purified via CombiFlash Rf (C18Aq 30 g, 35mL/min, CH₃CN/H₂O linear gradient from 0-100% CH₃CN over 15 minutes).The desired product (IIA4a) was isolated as a white powder (2.29 mg,0.06 mmol, 66%). Observed ESI HRMS m/z m/z=981.5106 [M+H]⁺.

A reaction chamber equipped with magnetic stirbar was charged withFmoc-pentapeptide (IIA4a, 2.9 mg, 2.96 μmol). The material was dissolvedin CH₃CN (1.0 mL) and purged with Ar. Diethylamine (0.401 mL, 3.84 mmol,1300 eq) was then added at rt and allowed to stir for 0.5 h. Thesolution was concentrated and purified using CombiFlash Rf (C18Aq 30 g,35 mL/min, CH₃CN/H₂O linear gradient from 0-100% CH₃CN over 15 minutes).The desired product was isolated as a white powder (1 mg, 1.317 μmol,45%). Observed ESI HRMS m/z m/z=380.2291 [M+2H]²⁺.

Synthesis of Compound 114:

A reaction chamber equipped with magnetic stirbar was charged with freebase tetrapeptide IIA2a (75 mg, 0.111 mmol) and 2-methylaminoisobutyricacid (13 mg, 0.111 mmol, 1 equiv.). The materials were dissolved inanhydrous DMF (1.5 mL) and cooled in an ice bath (−5° C.) under Arfollowed by addition of N,N-diisopropylamine (78 μL, 0.445 mmol, 4equiv.) and HATU (63 mg, 0.167 mmol, 1.5 equiv.). The resulting mixturewas allowed to stir for 90 min at 5° C. The mixture was then loaded ontoHP20ss (4 g) and purified via CombiFlash Rf (C18 15.5 g, 30 mL/min,CH₃CN/H₂O linear gradient from 15-100% CH₃CN over 14 minutes). Compounds107 (13 mg, 0.017 mmol, 15%) and 114 (11 mg, 0.014 mmol, 13%) were eachisolated as glassy amorphous solids. Observed ESI HRMS for compound 114:m/z 773.4696 [M+H]⁺; Compound 114 exists as two conformers in a 3:2ratio in CD30D solvent; the NMR chemical shifts for the major 114conformer are listed here. ¹H NMR (CD₃OD, 600 MHz): δ 7.79 (d, J=3.2 Hz,1H), 7.53 (d, J=3.2 Hz, 1H), 7.31 (d, J=7.3 Hz, 2H), 7.25 (t, J=7.3 Hz,2H), 7.21 (d, J=7.3 Hz, 1H), 5.66 (dd, J=4.1, 11.4 Hz, 1H), 5.02 (s,1H), 4.15 (m, 1H), 3.67 (m, 1H), 3.57 (m, 1H), 3.51 (dd, J=4.5, 14 Hz,1H), 3.42 (d, J=10.8 Hz, 1H), 3.38 (s, 3H), 3.35 (s, 3H), 3.33 (m, 1H),3.20 (m, 1H), 3.20 (s, 3H), 3.04 (m, 1H), 2.59 (s, 3H), 2.48 (m, 1H),2.38 (m, 1H), 2.29 (m, 1H), 1.89 (m, 1H), 1.88 (m, 1H), 1.71 (m, 1H),1.62 (s, 3H), 1.60 (m, 1H), 1.55 (s, 3H), 1.40 (m, 1H), 1.39 (s, 3H),1.37 (m, 1H), 1.26 (s, 3H), 1.16 (d, J=6.6 Hz, 3H), 1.04 (m, 1H), 1.01(d, J=7.3, 3H), 0.87 (t, J=7.4, 3H).

Example 2 General Procedure B—Synthesis of Reduced Antibody (Ab(r))

Purified antibody (Ab) may be buffer exchanged into a buffer such as PBS(pH 7.4) and then further diluted. Purified antibody (Ab) may be dilutedto a final concentration, for example, 5 mg/mL in buffer and warmed in aheat block. A stock solution of reducing agent such as TCEP(tris(2-carboxyethyl)phosphine) may be added to the antibody buffersolution to provide partially reduced antibody (Ab(r)).

General Procedure C—Synthesis of Antibody Drug Conjugate (ADC)

A stock solution of compound II-A in a solvent may be added to thepartially reduced antibody (Ab(r)) and allowed to mix for a period oftime to provide antibody drug conjugate compound III-A. After a periodof time, the mixture may be buffer exchanged and stored at reducedtemperature until needed. The drug to antibody ratio (DAR) may bemeasured by Hydrophobic Interaction Chromatography (HIC) and aggregationmay be measured by Size Exclusion Chromatography (SEC).

Example 2A: Synthesis of Conjugate 107-L¹-Ab1 and MMAD-L1-Ab1

Where R^(D) is Ab1-Mc-Val-Cit-PAB—C(O)—; Ab1=Antibody

The drug-Linker 107-L¹ and MMAD-L¹, which corresponds to compounds 107and MMAD respectively, where L¹ is Mc-Val-Cit-PAB-, were synthesizedusing protocols similar to methods described previously (Ref: Doronina,S. O. et al., Bioconjugate Chem. 2008, 19 (10), 1960-1963).

The antibody-drug conjugates 107-L¹-Ab1 and MMAD-L¹-Ab1 which correspondto drug-linkers 107-L¹ and MMAD-L¹ respectively, wherein Ab isundisclosed antibody, were synthesized using protocols similar tomethods described previously (Ref: Doronina, S. O. et al, BioconjugateChem. 2008, 19 (10), 1960-1963). Briefly, the purified antibody, Ab wasbuffer exchanged into PBS (pH 7.4). The antibody was diluted to a finalconcentration of 5 mg/mL in PBS and warmed to 37° C. in a heat block. Astock solution of TCEP (tris(2-carboxyethyl)phosphine, 50 mM) wasfreshly prepared in water, and 2.5 molar equivalent (relative to theantibody concentration) was added. After 2 h, the partially reducedantibody was removed from the heat block and cooled to room temperature.A stock solution of drug-linker 107-L¹ or MMAD-L¹ (2 mM in DMSO) wasfreshly prepared, and 2-5 molar equivalent was added to the antibody.After 1 h, the reaction mixture was buffer exchanged into PBS using PD10spin columns to remove small MW reagents and stored at 4° C. untilneeded. The drug to antibody ratio (DAR) was measured by HydrophobicInteraction Chromatography (HIC) and aggregation was measured by SizeExclusion Chromatography (SEC).

Example 3. Inhibitory Response of Test Compounds Against MES-SA andMES-SA/Dx Cells

MES-SA (human uterine sarcoma) cells were seeded in a clear polystyrene96-well microculture plate (Corning® Costar® 96-well flat bottom plate,Cat. #3997) in a total volume of 90 μL/well. After 24 hours ofincubation in a humidified incubator at 37° C. with 5% CO₂ and 95% air,10 μL of OX, serially diluted test agents in growth medium were added toeach well (10 pt dose response curve, highest concentration 10 μM oftest agent). After 72 hours of culture in a humidified incubator at 37°C., in an atmosphere of 5% CO₂ and 95% air, the plated cells and CellTiter-Glo® (Promega G7571) reagents were brought to room temperature toequilibrate for 30 minutes. 100 μL of Cell Titer-Glo® reagent was addedto each well. The plate was shaken for two minutes and then left toequilibrate for ten minutes. The media/Cell Titer-Glo® reagent wastransferred to a white polystyrene 96-well microculture plate (Corning®Costar® 96-well flat bottom plate, Cat. #3917) before readingluminescence on a Tecan GENios microplate reader.

MES-SA/Dx (multidrug-resistant human uterine sarcoma) cells are seededin a clear polystyrene 96-well microculture plate in a total volume of90 μL/well. After 24 hours of incubation in a humidified incubator at37° C. with 5% CO₂ and 95% air, 10 μL of 10×, serially diluted testagents in growth medium are added to each well. After 72 hours ofculture in a humidified incubator at 37° C., in an atmosphere of 5% CO₂and 95% air, the plated cells and Cell Titer-Glo® (Promega G7571)reagents are brought to room temperature to equilibrate for 30 minutes.100 μL of Cell Titer-Glo® reagent are added to each well. The plate isshaken for two minutes and then left to equilibrate for ten minutes. Themedia/Cell Titer-Glo® reagent is transferred to a polystyrene 96-wellmicroculture plate before reading luminescence on a Tecan GENiosmicroplate reader.

Percent inhibition of cell growth is calculated relative to untreatedcontrol wells. The IC₅₀ value for the test agents is determined usingPrism 6.05 by curve-fitting of the data using the following fourparameter-logistic equation:

${Y = {\frac{{Top} - {Bottom}}{1 + \left( {X/{IC}_{50}} \right)^{*}} + {Bottom}}},$

where Top is the maximal % of control absorbance, Bottom is the minimal% of control absorbance at the highest agent concentration, Y is the %of control absorbance, X is the agent concentration, IC₅₀ is theconcentration of agent that inhibits cell growth by 50% compared to thecontrol cells, and n is the slope of the curve. Data for compounds 101,109 and 110 is shown in Table 3.

TABLE 3 MES SA MES DX Compound IC₅₀ in nM IC₅₀ in nM 101 0.03496 6.373109 14.9 NT 110 4.185 NT

Example 4: Inhibitory Response of Test Compounds Against BT-474 Cells

BT-474 human mammary gland ductal carcinoma cells were seeded in a clearpolystyrene 96-well microculture plate (Corning® Costar® 96-well flatbottom plate, Cat. #3997) in a total volume of 90 μL/well. After 24hours of incubation in a humidified incubator at 37° C. with 5% CO₂ and95% air, 10 μL of OX, serially diluted test agents in growth medium wereadded to each well (10 pt dose response curve, highest concentration 10μM of test agent). After 72 hours of culture in a humidified incubatorat 37° C., in an atmosphere of 5% CO₂ and 95% air, the plated cells andCell Titer-Glo® (Promega G7571) reagents were brought to roomtemperature to equilibrate for 30 minutes. 100 μL of Cell Titer-Glo®reagent was added to each well. The plate was shaken for two minutes andthen left to equilibrate for ten minutes. The media/Cell Titer-Glo®reagent was transferred to a white polystyrene 96-well microcultureplate (Corning® Costar® 96-well flat bottom plate, Cat. #3917) beforereading luminescence on a Tecan GENios microplate reader.

Percent inhibition of cell growth was calculated relative to untreatedcontrol wells. The IC₅₀ value for the test agents was determined usingPrism 6.05 by curve-fitting of the data using the following fourparameter-logistic equation:

${Y = {\frac{{Top} - {Bottom}}{1 + \left( {X/{IC}_{50}} \right)^{*}} + {Bottom}}},$

where Top is the maximal % of control absorbance, Bottom is the minimal% of control absorbance at the highest agent concentration, Y is the %of control absorbance, X is the agent concentration, IC₅₀ is theconcentration of agent that inhibits cell growth by 50% compared to thecontrol cells, and n is the slope of the curve.

Inhibitory response of test compounds against HCC1954 cells wasdetermined using a method analogous to that used for BT-474 cells. IC₅₀values for various test agents in HCC1954 and/or BT-474 cells are shownin Table 4.

TABLE 4 BT-474 HCC1954 Compound IC₅₀ in nM IC₅₀ in nM 101 0.0022 0.004104 0.364 0.435 105 8.554 7.966 106 2.369 1.137 107 0.064 0.030 1080.140 0.460 109 26.10 10.92 110 9.088 2.924 114 0.0616 0.017 111 0.86340.06556 112 NT NT 113 1.221 1.235

Example 5: Inhibitory Response of Test Conjugates Against 293T Cells

293T human embryonic kidney cells were seeded in a clear polystyrene96-well microculture plate in a total volume of 90 μL/well. After 24hours of incubation in a humidified incubator at 37° C. with 5% CO₂ and95% air, 10 μL of OX, serially diluted test agents (e.g., conjugates,such as compounds bound to an antibody via a linker) in growth mediumwere added to each well. After 72 hours of culture in a humidifiedincubator at 37° C., in an atmosphere of 5% CO₂ and 95% air, the platedcells and Cell Titer-Glo® (Promega G7571) reagents were brought to roomtemperature to equilibrate for 30 minutes. 100 μL of Cell Titer-Glo®reagent were added to each well. The plate was shaken for two minutesand then left to equilibrate for ten minutes. The media/Cell Titer-Glo®reagent was transferred to a polystyrene 96-well microculture platebefore reading luminescence on a Tecan GENios microplate reader.

Percent inhibition of cell growth was calculated relative to untreatedcontrol wells. The IC₅₀ value for the test agents was determined usingPrism 6.05 by curve-fitting of the data using the following fourparameter-logistic equation:

${Y = {\frac{{Top} - {Bottom}}{1 + \left( {X/{IC}_{50}} \right)^{*}} + {Bottom}}},$

where Top is the maximal % of control absorbance, Bottom is the minimal% of control absorbance at the highest agent concentration, Y is the %of control absorbance, X is the agent concentration, IC₅₀ is theconcentration of agent that inhibits cell growth by 50% compared to thecontrol cells, and n is the slope of the curve. Data for antibody-drugconjugates tested is shown in Table 5.

TABLE 5 % MES-SA 293T Test Agent DAR Aggregate IC₅₀ (nM) IC₅₀ (nM)107-L1-Ab1 3.8 2.4 3293 0.079 MMAD-L1-Ab1 3.6 12.6 NT 0.012

While the foregoing written description of the compounds, uses, andmethods described herein enables one of ordinary skill to make and usethe compounds, uses, and methods described herein, those of ordinaryskill will understand and appreciate the existence of variations,combinations, and equivalents of the specific embodiment, method, andexamples herein. The compounds, uses, and methods provided herein shouldtherefore not be limited by the above-described embodiments, methods, orexamples, but rather encompasses all embodiments and methods within thescope and spirit of the compounds, uses, and methods provided herein.

All references disclosed herein are incorporated by reference in theirentirety.

While one or more embodiments of the present disclosure have beendescribed, it will be understood by those of ordinary skill in the artthat various changes in form and details may be made therein withoutdeparting from the spirit and scope of the present embodiments asdefined by the following claims.

What is claimed is:
 1. A compound having the structure of Formula IIa,IIb, IIc or IId:

or pharmaceutically acceptable salts or solvates thereof, wherein: R¹ isselected from the group consisting of hydrogen, deuterium, an optionallysubstituted C₁-C₆ alkyl, an optionally substituted C₃-C₇ cycloalkyl, anoptionally substituted aryl, and an optionally substituted heteroaryl;R² is selected from the group consisting of R^(D), an optionallysubstituted C₁-C₆ alkyl, an optionally substituted C₃-C₇ cycloalkyl, anoptionally substituted aryl, and an optionally substituted heteroaryl;R³ is selected from the group consisting of hydrogen, deuterium, andoptionally substituted C₁-C₆ alkyl, an optionally substituted hydroxyl,an optionally substituted C₁-C₆ alkoxy, an optionally substituted C₃-C₇cycloalkyl, an optionally substituted aryl, and an optionallysubstituted heteroaryl; R⁴ is an optionally substituted C₁-C₆ alkyl; R⁵is hydrogen, deuterium or an optionally substituted C₁-C₆ alkyl; R⁶ ishydrogen, deuterium or an optionally substituted C₁-C₆ alkyl; X¹ and X²are each independently selected from the group consisting of hydrogen,deuterium, an optionally substituted C₁-C₆ alkyl, an optionallysubstituted aryl, an optionally substituted heteroaryl, halogen, —CN,—N₃, —COOR^(B), —NR^(A)R^(B), —OR^(B), SR^(B), —COOH, —NHR^(A), —OH, and—SH; wherein at least one of X¹ and X² in Formula IIa and Formula IId isselected from the group consisting of an optionally substituted aryl, anoptionally substituted heteroaryl, halogen, —CN, —N₃, —COOR^(B),—NR^(A)R^(B), OR^(B), SR^(B), —COOH, —NHR^(A), —OH, and —SH; R^(A) isselected from the group consisting of hydrogen, deuterium, an optionallysubstituted C₁-C₆ alkyl, an optionally substituted C₃-C₇ cycloalkyl, anoptionally substituted aryl, and an optionally substituted heteroaryl;R^(B) is selected from the group consisting of R^(1A)-L¹-, R^(3A)-L¹-,R^(4A)-L¹-, Mc-L¹-, Mal-L³-L¹-, R^(1A)-Mal-L³-L¹-Val-Cit-PAB—C(O)— orMal-L³-L¹-Val-Cit-PAB—C(O)—; R⁷ is an optionally substituted C₁-C₆alkyl, an optionally substituted heteroaryl, or —C(═O)R^(C); R^(C) isselected from the group consisting of an optionally substituted C₁-C₆alkyl, optionally substituted C₁-C₆ alkoxy, an optionally substitutedC₃-C₇ cycloalkyl, an optionally substituted aryl, and an optionallysubstituted heteroaryl, and hydroxyl; and R⁸ is hydrogen, deuterium,—OR^(D), or —SR^(D); R^(D) is selected from the group consisting ofR^(1A)-L¹-, R^(3A)-L¹-, R^(4A)-L¹-, Mc-L¹-, Mal-L³-L¹-,R^(1A)-Mal-L³-L¹-Val-Cit-PAB—C(O)— and Mal-L³-L¹-Val-Cit-PAB—C(O)—;R^(3A) is

R^(4A) is a conjugation moiety; R^(1A) is a conjugated targeting moiety;L¹ is a linker or a bond; and L³ is an alkanoyl.
 2. The compound ofclaim 1, wherein R^(D) is R^(1A)-Mc-Val-Cit-PAB—C(O)—,R^(1A)-Mal-L³-Val-Cit-PAB—C(O)—, Mc-Val-Cit-PAB—C(O)— orMal-L³-Val-Cit-PAB—C(O)—.
 3. The compound of claim 1, wherein R^(B) isR^(1A)-Mc-Val-Cit-PAB—C(O)—, R^(1A)-Mal-L³-Val-Cit-PAB—C(O)—,R^(1A)-Mal-L³-Val-Cit-PAB—C(O)—, Mc-Val-Cit-PAB—C(O)— orMal-L³-Val-Cit-PAB—C(O)—.
 4. The compound of claim 1, wherein R⁸ ishydrogen, or —OR^(D).
 5. The compound of claim 1, wherein X¹ ishydrogen; and X² is —OR^(B), or X² is hydrogen; and X¹ is —OR^(B). 6.The compound of claim 1, wherein the targeting moiety binds to one ormore tumor-associated antigens or cell surface receptors selected fromthe group consisting of CD19, CD22, CD30, CD33, CD56, CD70, CD79b, CD74,CD138, HER2, GPNMB, PSMA, SLC44A4, CA6, CA-IX, Mesothelin, CD66e,CEACAM5, and Nectin-4, or the targeting moiety is a protein ligand, aprotein scaffold, a peptide, cysteine-engineered antibody, antibody-likeprotein, monoclonal antibody (mAB), an antibody fragment, surrogate, orvariant.
 7. The compound of claim 1, wherein R^(1A) comprises atargeting moiety selected from the group consisting of brentuximab,inotuzumab, gemtuzumab, milatuzumab, trastuzumab, glembatumomab,lorvotuzumab, or labestuzumab, or derivatives thereof.
 8. The compoundof claim 1, wherein L¹ is—(CHR¹³)—CH₂—(CR¹⁴R¹⁵)—S—S—(CR¹⁶R¹⁷)—(CH₂)_(n)(CO)_(r)—; n is 1, 2, 3,4, or 5; r is 0 or 1; R¹³ is hydrogen or SO₃H; and R¹⁴, R¹⁵, R¹⁶ and R¹⁷are each independently hydrogen or an optionally substituted C₁-C₆alkyl.
 9. The compound of claim 1, wherein L¹ comprises

or a dipeptide selected from the group consisting of -Phe-Lys-,-Val-Ala-, -Val-Lys-; -Ala-Lys-, -Val-Cit-, -Phe-Cit-, -Leu-Cit-,-Ile-Cit-, -Phe-Arg-, and -Trp-Cit-.
 10. The compound of claim 1,wherein R^(1A)-L¹- is

where q is 0 to 6; and L is a linker, or R^(1A)-L¹- is

where s is 0 or 1; and t is 0 to 30, or R^(1A)-L¹- is

where t is 0 to
 30. 11. The compound of claim 1, wherein: R^(4A)— is

and R is hydrogen, —C(═O)N(CH₂CH₃)₂, or —SO₂N(CH₂CH₂)₂O.
 12. Thecompound of claim 1, wherein R^(D) is


13. The compound of claim 1, wherein R^(B) is


14. The compound of claim 1, wherein the compound is

or a stereoisomer or pharmaceutically acceptable salt thereof, whereinR^(D) is Ab1-Mc-Val-Cit-PAB—C(O)— and Ab1 is an antibody.
 15. A methodof treating cancer, comprising administering a compound of claim 1 to asubject in need thereof, wherein the cancer is selected from the groupconsisting of a carcinoma, a sarcoma, a lymphoma, and a blastoma.
 16. Amethod of treating cancer, comprising administering a compound of claim1 to a subject in need thereof, wherein the cancer is selected from thegroup consisting of uterine sarcoma cancer, bladder cancer, breastcancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer,lung cancer, melanoma, non-Hodgkin lymphoma, glioblastoma, pancreaticcancer, prostate cancer, and thyroid cancer.
 17. A pharmaceuticalcomposition comprising a compound of claim 1 and a pharmaceuticallyacceptable excipient.